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【摘要】 Fetuin-A is a known inhibitor of vascular calcification in vitro. In arteries with calcification, there is increased immunostaining for fetuin-A. However, vascular smooth muscle cells (VSMC) do not synthesize fetuin-A, suggesting fetuin-A may be endocytosed to exert its inhibitory effects. To examine the mechanism by which fetuin-A is taken up in bovine VSMC (BVSMC), we examined living cells by confocal microscopy and determined the uptake of Cy5-labeled fetuin-A. The results demonstrated that fetuin-A was taken up in BVSMC only in the presence of extracellular calcium, whereas phosphorus had no effect. Additional studies demonstrated the calcium-dependent uptake was specific for fetuin-A and only observed in BVSMC and osteoblasts, but not epithelial, endothelial, or adipose cells. The uptake was dose dependent, but could not be inhibited by excess unlabeled fetuin-A, suggesting a fluid phase rather than a receptor-mediated process. Fetuin-A also induced a sustained increase in intracellular calcium in BVSMC in the presence of extracellular calcium, whereas there was no increase in the absence of extracellular calcium. To further characterize the uptake, we utilized an inhibitor of annexin calcium channel activity, demonstrating inhibition of both fetuin-A uptake and intracellular calcium increase. Finally, we demonstrate that fetuin-A binds to annexin II at the cell membrane of BVSMC. In summary, our study demonstrates calcium- and annexin-dependent uptake of fetuin-A that leads to a sustained rise in intracellular calcium. This regulated uptake may be a mechanism by which fetuin-A inhibits VSMC calcification in the presence of excess calcium.
【关键词】 annexins cellular uptake intracellular calcium vascular calcification
VASCULAR CALCIFICATION IS common in aging, diabetes, and kidney disease and is associated with increased cardiovascular morbidity and mortality ( 18, 19, 23 ). Risk factors vary in these patient groups, but advanced age and inflammation are commonly associated with increased vascular calcification. Recent attention has focused on fetuin-A, a reverse acute-phase protein, with levels inversely related to C-reactive protein and proinflammatory cytokine serum levels. Fetuin-A ( -2-Heremens Schmidt glycoprotein) is an abundant serum glycoprotein of 65,000 Da that is a member of the cystatin superfamily of proteins ( 25 ). Ketteler et al. ( 13 ) found that low levels of fetuin-A are associated with cardiovascular mortality in dialysis patients, data confirmed by two other groups ( 26, 30 ). Low fetuin-A levels in dialysis patients are associated with increased coronary artery calcification by spiral computed tomography scan ( 20 ), valvular calcification ( 30 ), and increased intima medial thickening of the carotid arteries ( 26 ). Fetuin-A knockout animals have diffuse extraosseous calcification ( 24 ), indicating an important role for fetuin-A in the inhibition of unwanted mineralization. Fetuin-A binds to calcium and phosphorus to create small particles in serum, accounting for 50% of the effect of serum to inhibit calcium-phosphate precipitation ( 10 ). These data support a potential role for fetuin-A in the regulation of extraskeletal calcification.
Fetuin-A also appears to have a direct cellular inhibitory effect on vascular calcification. Fetuin-A can decrease calcification of osteoblasts ( 10 ) and vascular smooth muscle cell (VSMC) in vitro ( 20, 22 ). We have also found that fetuin-A was present by immunostaining in areas of atherosclerosis and medial calcification in the inferior epigastric artery of chronic kidney disease patients ( 20 ). Since VSMC do not synthesize fetuin-A ( 28 ), this implies that the fetuin-A deposits there in an attempt to limit calcification. Reynolds et al. ( 22 ) recently demonstrated that, when fetuin-A was present in matrix vesicles from VSMC, they were rendered less capable of mineralization, suggesting that the cellular uptake of fetuin-A may have intracellular effects on the development of matrix vesicles to inhibit mineralization. However, the mechanism by which VSMC may take up fetuin-A is unknown.
A recent study in a breast carcinoma cell line demonstrated that membrane expressed annexin II and VI bind to immobilized fetuin-A in a calcium-dependent manner ( 17 ). Annexins are a family of closely related calcium and membrane binding proteins expressed in most eukaryotic cell types with diverse functions, including vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation. In addition, several annexins, in particular annexins I, II, and VI, are present on endosomal compartments and are involved in endocytosis ( 6 ). In vitro, calcium entry into chondrocytes upregulates annexins II, V, and VI, which in turn mediate calcium influx into matrix vesicles ( 31, 32 ). Furthermore, mineralization of chondrocyte matrix vesicles is inhibited by antibodies to annexins II and VI ( 16 ). The purpose of the present study was to determine the mechanism of fetuin-A uptake by bovine VSMC (BVSMC) and the role of annexins in this cellular uptake.
METHODS
Cell culture. BVSMC were isolated from thoracic aorta by the explant method, as previously described ( 3 ). The BVSMC were grown in Dulbecco's modified Eagles medium (DMEM; Sigma, St. Louis, MO), with 10% FBS until confluent, at which time they were reseeded for specific experiments. In some experiments, BVSMC were exposed to serum-free media for 24 h before the experiments. Only cells between passages 2 and 8 are used in the experiments. These cells express -smooth muscle actin, as well as basal alkaline phosphatase, and Runx2 (Cbfa-1) ( 2 ).
Fluorescent labeling of fetuin and live cell imaging. To determine the mechanisms of BVSMC uptake of fetuin-A, we examined living cells using confocal microscopy. Fetuin-A from fetal calf serum (Sigma) was labeled with FluoroLink Cy5 mono-functional dye (Amersham Biosciences, Piscataway, NJ). It was separated on a column of Bio-Gel P-30 Gel (Bio-Rad Laboratories, Hercules, CA). Cy-5-labeled bovine albumin of similar size (66 kDa, Sigma, St. Louis, MO) was used as a protein control. Dextran-Texas Red of similar size (70 kDa; Molecular Probes, Eugene, OR) was also used as a control for the endocytic process. In some experiments, Cy-5-labeled asialofetuin-A was also used. To determine cellular specificity, we also examined uptake of fluorescently labeled fetuin-A in both mineralizing and nonmineralizing cells, including MC3T3 osteoblast cells, Madin-Darby canine kidney (MDCK) renal epithelial cells, human aortic endothelial cells, and 3T3-L1 adipocytes (gifts from Drs. R. Duncan, R. Bacallao, K. March, and R. Considine, Indiana University). BVSMC and these other cells were seeded on glass bottom microwell dishes (MatTek, Ashland, MA) in 10% FBS DMEM for 72 h. Ten minutes before addition of Cy5-fetuin, the media was replaced with M2 media (150 mM NaCl, 20 mM HEPES, 0 or 1.3 mM CaCl 2, 5 mM KCl, 1 mM MgCl 2, 50 mM glucose, at pH 7.4) at 37°C. Labeled fetuin-A was then added, and a MRC-1024 laser scanning confocal microscopy (Bio-Rad) was used to capture images. Plates were kept at a constant 37°C.
To determine the effect of calcium and phosphorus on the fetuin-A uptake, BVSMC were incubated with M2 media with 0, 1.3, and 5 mM CaCl 2, with or without various concentrations of NaH 2 PO 4. BVSMC were also pretreated with various doses of K201 ( 11 ), a specific annexin calcium channel blocker (kindly provided by Aetas Pharma) for 24 h before switching to test media. In some experiments, BVSMC were pretreated with 30 µM BAPTA-AM, an intracellular calcium chelator (Calbiochem), or 10 µM nifedipine (selective blocker of L-type Ca 2+ channel, Calbiochem) for 30 min before live cell imaging of fetuin-A uptake experiment. Each experiment was repeated three to six times, with at least two dishes per experiment for a total n of 6-12. To quantify the uptake of fluorescently labeled fetuin-A, image processing was conducted using Metamorph software (Universal Imaging, West Chester, PA). Six images with four to six cells per image were quantified to allow for a representative assessment of the uptake.
Western blotting. Western blotting was performed as previously described ( 3 ). The blots were incubated with rabbit antibodies against annexin II (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA) or bovine fetuin-A (1:2,000, a gift from Dr. Willi Jahnen-Dechent, Aachen, Germany) overnight at 4°C followed by incubating with peroxidase-conjugated secondary antibody (1:5,000 dilution). Immunodetection was with the Enhanced Chemiluminescence Kit (Amersham, Piscataway, NJ). The band intensity was analyzed by scanning densitometry (Quantity One, Bio-Rad, Richmond, CA).
Intracellular Ca 2+ measurement. To determine the effects of fetuin-A on cytosolic free Ca 2+ concentration ([Ca 2+ ] i ), BVSMC were grown on a glass coverslip in DMEM with 10% FBS. The cells were then switched to serum-free media for 24 h to minimize residual effects of fetuin-A. Before the analysis for [Ca 2+ ] i, cells were loaded with 3 µM fura 2-AM (Molecular Probes, Eugene, OR), a fluorescent [Ca 2+ ] i chelator, in Hanks' balanced saline solution for 30 min at 37°C. Cells were rinsed and incubated for an additional 30 min with Hanks' balanced saline solution alone to allow for complete deesterification of the fluorescent probe. A ratiometric video-image analysis apparatus (Intracellular Imaging, Cincinnati, OH) was used to determine changes in [Ca 2+ ] i. The ratio of consecutive frames obtained at 340 and 380 nm is determined, and the [Ca 2+ ] i in each cell is calculated from this ratio by comparison to a fura 2 free acid standard curve, as previously described ( 4 ). The net [Ca 2+ ] i response was calculated by determining the percent increase of peak calcium levels over baseline in response to the addition of fetuin-A in the presence or absence of 10 µM K201 in 0 or 1.3 mM CaCl 2. Bovine serum albumin was used as a control for nonspecific findings. In some experiments, 10 µM ionomycin was used as positive control for [Ca 2+ ] i measurement.
Plasma membrane protein preparation and immunoprecipitation. BVSMC were incubated in serum-free media in the presence or absence of high calcium and phosphorus concentrations (2.6 mM CaCl 2, 2.0 mM NaH 2 PO 4 = high Ca x P i ), normal calcium and phosphorus concentrations (1.8 mM CaCl 2, 0.9 mM NaH 2 PO 4 = normal Ca x P i ), with or without fetuin-A (500 µg/ml) and cell membrane fractions of BVSMC isolated using the ProteoPrep Membrane Extraction Kit, according to kit instructions (Sigma Chemical). To analyze the interaction between annexins and fetuin-A, 500 µg of plasma membrane protein were immunoprecipitated with rabbit antibody against fetuin-A in the presence of ExactaCruz immunoprecipitation reagent (Santa Cruz Biotechnology). The pellet was washed with PBS and resuspended in 2 x reducing buffer, boiled, and loaded onto a 10% SDS-PAGE. Western blotting was then performed as described above with antibody against annexin II.
Statistical analysis. The difference in quantified fetuin-A uptake by live cell imaging, bands assessed by densitometry on Western blot, or percent change in intracellular calcium in BVSMC in response to various treatments were compared by ANOVA with Fisher's post hoc analysis. The results are expressed as means ± SD, with P < 0.05 considered significant (StatView, SAS Institute, Cary, NC).
RESULTS
Fetuin-A is endocytosed by BVSMC in a calcium-dependent manner. To confirm that fetuin-A was endocytosed, BVSMC were incubated with 0, 1.3 (normal), or 5 mM CaCl 2 (high calcium) in the presence of Cy5-labeled fetuin-A (100 µg/ml) and examined using confocal microscopy, with the magnitude of fetuin-A uptake quantified by MetaMorph software. We determined that fetuin-A was endocytosed by BVSMC after 25-35 min, and that this was a calcium-dependent process ( Fig. 1, A and B ). As shown in Fig. 1 C, there is minimal fetuin-A uptake in BVSMC when there is no extracellular calcium. Quantitative analysis showed a 4.7-fold increases in fetuin-A uptake in the presence of normal (1.3 mM) extracellular calcium compared with 0 mM calcium ( P < 0.05). However, there is no difference in fetuin-A uptake between normal (1.3 mM) or high (5 mM) extracellular calcium concentrations ( Fig. 1 C ). To confirm the live cell imaging uptake of fetuin-A, BVSMC were also incubated with or without fetuin-A (100 µg/ml) in the presence of various calcium concentrations after serum starvation for 24 h, and cell lysate was isolated for Western blot of fetuin-A ( Fig. 1 D ). The results confirm the live cell imaging experiments, demonstrating cellular fetuin-A uptake is dependent on extracellular calcium. Since BVSMC were incubated with 10% FBS presumably containing relatively high amounts of fetuin-A, we performed the additional experiments in which BVSMC were exposed to serum-free media for 24 h before labeled fetuin-A was added for live cell imaging. The results demonstrated that, while the fetuin-A uptake occurred earlier (30 min after adding labeled fetuin-A) in BVSMC with overnight serum starvation compared with BVSMC without serum starvation (40 min after adding labeled fetuin-A), the uptake reached the same degree by 60 min (data not shown). Furthermore, exposure of BVSMC to serum-free media for 24 h did not change the calcium-dependent pattern of fetuin uptake.
Fig. 1. Fetuin-A is endocytosed by bovine vascular smooth muscle cell (BVSMC) in a calcium-dependent manner. BVSMC were incubated with 0 ( A ), 1.3 mM ( B ), or 5 mM CaCl 2 (high calcium) in the presence of Cy5-labeled fetuin-A (100 µg/ml), and fetuin-A uptake was examined by confocal microscopy. The degree of fetuin-A uptake was also quantified by MetaMorph software (intensity/cell). C : the results demonstrated that fetuin-A uptake by BVSMC is calcium dependent. Data are shown as means ± SD from four experiments. * P < 0.05. D : BVSMC were also incubated with various calcium concentrations in the presence of 100 µg/ml fetuin-A for 30 min, and cell lysate was isolated for Western blot of fetuin-A.
To characterize the role of phosphorus in fetuin-A uptake, BVSMC were incubated with increasing concentrations (0, 1.8, or 5 mM) of NaH 2 PO 4 in the presence or absence of normal (1.3 mM) calcium, and fetuin-A uptake was examined by confocal microscopy and quantified. The results demonstrated that, in the absence of extracellular calcium, phosphorus alone had no effect on fetuin-A uptake in BVSMC ( Fig. 2 A ). Furthermore, in the presence of 1.3 mM extracellular calcium, there is no difference in fetuin-A uptake with increasing concentrations of phosphorus ( Fig. 2 B ). These data indicate that phosphorus had no additive effect on the calcium-dependent fetuin-A uptake in BVSMC ( Fig. 2 B ).
Fig. 2. The role of phosphorus in fetuin-A uptake in BVSMC. BVSMC were incubated with 0, 1.8, or 5 mM NaH 2 PO 4 (P i ) in the presence of 0 or 1.3 mM CaCl 2 (Ca 2+ ). Cy5-labeled fetuin-A (100 µg/ml) was added to the cells, and uptake was examined by confocal microscopy, and the degree of fetuin-A uptake was quantified (intensity/cell). A : the results demonstrated that there is no or minimal fetuin-A uptake by BVSMC incubated with various concentrations of phosphorus in the absence of extracellular calcium. B : in the presence of normal calcium (1.3 mM Ca 2+ ), there is no significant difference in fetuin-A uptake in BVSMC treated with various concentrations of phosphorus, suggesting that phosphorus has no additional effect on fetuin-A uptake.
To further evaluate the calcium-dependent uptake of fetuin-A in BVSMC, we performed additional experiments. First, to determine whether the calcium dependency of fetuin-A uptake is specific for fetuin-A, uptake of Cy-5-labeled bovine albumin and Texas-red-labeled dextran of similar molecular weight as fetuin-A were examined in the absence or presence of extracellular calcium concentration. The results demonstrated that fluorescently labeled fetuin-A dextran and albumin were all taken up by the BVSMC, but only the uptake of fetuin-A was calcium dependent. The albumin and dextran uptake in BVSMC was independent of extracellular calcium (data not shown). Second, to determine the cell specificity of fetuin-A uptake, we examined the uptake of Cy5-fetuin-A, with and without 1.3 mM calcium, in MDCK renal epithelial cells, human aortic endothelial cells, 3T3-L1 adipocytes, and MC3T3 osteoblasts. The results demonstrated that a similar calcium-dependent uptake was observed only in MC3T3 osteoblasts. Fetuin-A was also taken up, but in a non-calcium-dependent manner, in MDCK cells, endothelial cells, and adipocytes ( Fig. 3 ). These results suggest that calcium dependency of fetuin-A uptake is unique for BVSMC and MC3T3 osteoblasts. Interestingly, these are also the only two cell types that can also mineralize in vitro in the presence of calcium and phosphorus. To determine the mechanism by which BVSMC take up fetuin-A, cells were incubated with increasing doses of Cy5-labeled fetuin-A (25, 50, or 100 µg/ml), and uptake was examined by confocal microscopy and quantified. As demonstrated in Fig. 4 A, there is a significant dose-dependent uptake of fetuin-A in BVSMC ( P < 0.05). BVSMC were also incubated with Cy5-labeled fetuin-A in the presence or absence of 20-fold unlabeled fetuin-A and examined by confocal microscopy. The results demonstrated that excess unlabeled fetuin-A had no effect on Cy5-labeled fetuin-A uptake ( Fig. 4 B ). The finding of a dose-dependent uptake, together with an inability of excess fetuin-A to block uptake, suggests the mechanism of endocytosis is fluid phase rather than receptor mediated. Interestingly, asialofetuin-A, like other asialo-glycoprotein, is bound by the asialo-glycoprotein receptor on the cell surface and internalized ( 29 ). Indeed, we have found that excess unlabeled (20-fold) asialofetuin-A can partially block Cy5-labeled asialofetuin-A uptake in BVSMC (data not shown), suggesting a receptor-mediated process for asialofetuin-A uptake.
Fig. 3. Cell specificity of calcium-dependent fetuin-A uptake. To determine whether the calcium-dependent uptake of fetuin-A is specific for BVSMC, Madin-Darby canine kidney (MDCK), human aortic endothelial cells (HAEC), 3T3 adipocytes, and MC3T3 osteoblasts were used, and live-cell imaging was performed in M2 media with Cy5-labeled fetuin-A in the presence of 0 or 1.3 mM CaCl 2. The results demonstrate that osteoblasts endocytose fetuin-A in a calcium-dependent manner, similar to BVSMC. However, MDCK, HAEC, and adipocytes endocytose fetuin-A in a non-calcium-dependent manner.
Fig. 4. Mechanism of fetuin-A uptake in BVSMC. Receptor mediated vs. fluid phase transport. To determine whether there is a dose-dependent uptake of fetuin-A in BVSMC, live cell imaging was performed in M2 media with increasing doses of Cy5-labeled fetuin-A (25, 50, or 100 µg/ml) and quantified using MetaMorph software. A : the results demonstrated that there is a significant dose-dependent uptake of fetuin-A in BVSMC. B : however, this uptake of Cy5-labeled fetuin-A could not be inhibited by 20-fold unlabeled fetuin-A, suggesting that fetuin-A uptake is a fluid phase rather than receptor-mediated process. Data are shown as means ± SD from three experiments. * P < 0.05 compared with 25 µg/ml of Cy5-fetuin-A. # P < 0.05 compared with 50 µg/ml of Cy5-fetuin-A.
Fetuin-A increases intracellular calcium in BVSMC. To determine whether calcium-dependent fetuin-A uptake in BVSMC is associated with an alteration in intracellular calcium levels, we measured [Ca 2+ ] i using fura 2 by ration fluorescence spectrometer. In the presence of normal extracellular calcium (1.3 mM CaCl 2 ), the addition of fetuin-A (1 mg/ml) induced a sustained increase in [Ca 2+ ] i in BVSMC ( Fig. 5 A ). However, in the absence of extracellular calcium (0 mM CaCl 2 ), there was no increase in intracellular calcium in BVSMC when fetuin-A was added ( Fig. 5 B ). Addition of bovine serum albumin to the BVSMC had no effect on intracellular calcium levels (data not shown). To ensure the addition of fetuin-A with or without calcium did not injure the cells, 10 µM of the calcium ionophore ionomycin was added after the fetuin-A. As shown in Fig. 5, there was still a rapid and appropriate increase in [Ca 2+ ] i in BVSMC, indicating that the fetuin-A did not alter normal intracellular calcium responsiveness.
Fig. 5. Fetuin-A induces a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) in BVSMC. Fetuin-A (1 mg/ml) was added to BVSMC in the presence ( A ) or absence ( B ) of normal extracellular calcium (1.3 mM CaCl 2 ), and [Ca 2+ ] i was measured using fura 2 by a ratiometric video-imaging analysis. There was a sustained increase in [Ca 2+ ] i in the presence of extracellular calcium in BVSMC treated with fetuin-A ( A ), whereas there is no increase in [Ca 2+ ] i in the absence of extracellular calcium ( B ). A and B : to ensure this prolonged increase in Ca 2+ concentration was not due to cell damage, 10 µM ionomycin were added after fetuin-A was added, and there was still a rapid increase in Ca 2+ concentration in BVSMC.
Blocking annexin calcium channel activity prevents fetuin-A uptake and rise in intracellular calcium in BVSMC. To determine the role of annexins in the calcium-dependent uptake of fetuin-A in BVSMC, cells were pretreated with increasing concentrations of K201, an inhibitor of annexin Ca 2+ channel activity ( 11, 14 ) for 24 h, and fetuin-A uptake was examined by confocal microscopy and quantified. The results demonstrate that K201 dose-dependently inhibited calcium-dependent fetuin-A uptake in BVSMC ( Fig. 6 A; 34% inhibition in fetuin-A uptake with 5 µM K201; 70% inhibition in fetuin-A uptake with 10 µM K201; * P < 0.05 compared with 0 µM K201, # P < 0.05 compared with 5 µM K201, n = 6). However, treatment of BVSMC with BAPTA-AM, an intracellular calcium chealator, or nifedipine, a selective blocker of L-type Ca 2+ channel, had no effect on fetuin-A uptake in BVSMC (data not shown). These data suggest that annexins mediate the calcium-dependent uptake of fetuin-A in BVSMC.
Fig. 6. Blocking annexin calcium channel activity prevents fetuin-A uptake and rise in intracellular calcium in BVSMC. A : BVSMC were pretreated with increasing concentrations of K201, an inhibitor of annexin Ca 2+ channel activity for 24 h, and fetuin-A uptake was examined by confocal microscopy and quantified. The results demonstrate that K201 dose-dependently inhibited calcium-dependent fetuin-A uptake in BVSMC. * P < 0.05 compared with 0 µM K201; # P < 0.05 compared with 5 µM K201, n = 6. B : BVSMC were pretreated with or without 10 µM K201 for 24 h, and [Ca 2+ ] i in response to fetuin-A was examined. 10 µM ionomycin were used as control for [Ca 2+ ] i measurement. The results demonstrated that K201 treatment completely blocked fetuin-A-induced increase in [Ca 2+ ] i but had no effect on inonmycin-induced increase in [Ca 2+ ] i. * P < 0.05 compared with no fetuin-A; # P < 0.05 compared fetuin-A; n = 5.
To determine the role of annexins in fetuin-A-induced changes in intracellular calcium, BVSMC were pretreated with K201 for 24 h, and intracellular calcium was examined. As shown in Fig. 6 B, while fetuin-A increased [Ca 2+ ] i by 25 ± 9.5%, K201 treatment completely blocked fetuin-A-induced increase in intracellular calcium in BVSMC (0.8 ± 0.4%, P < 0.05). However, K201 treatment had no significant effect on ionomycin-induced increase in [Ca 2+ ] i (43 ± 10% increase for control cells treated with ionomycin alone; 49 ± 11% increase for K201 pretreated cells with ionomycin). These results support that, in BVSMC, annexins mediate the calcium-dependent uptake of fetuin-A and the sustained rise in [Ca 2+ ] i in response to fetuin-A.
Fetuin-A binds to annexin II in BVSMC. To characterize the interaction of fetuin-A and annexins further, we performed two sets of experiments. First, to determine whether fetuin-A increased the membrane location of annexin, BVSMC were treated with normal Ca x P i (1.3 mM Ca/1.4 mM P i ) or high Ca x P i (2.6 mM Ca/2.0 mM P i ) in the presence or absence of fetuin-A (500 µg/ml), and membrane fractions were isolated. Western blot analysis of membrane proteins was performed to examine the existence of annexin II in the cell membrane. As shown in Fig. 7 A, annexin II is present in the membrane of BVSMC, but neither high Ca x P i concentrations or fetuin-A changed the quantity of annexin II in membrane fraction of BVSMC. Second, to determine whether there is binding of fetuin-A to annexin II at the cell membrane, BVSMC were incubated with high Ca x P i or normal Ca x P i to see if conditions conducive to mineralization changed their binding. Membrane fractions were isolated and then immunoprecipitated with antibody against fetuin-A to precipitate membrane fractions that contained fetuin-A. These fetuin-A containing fractions were then analyzed to determine whether they also contained annexin II Western blot. The results showed that fetuin-A-containing membrane fractions from BVSMC treated with high Ca x P i had significantly more annexin II, whereas the membrane fraction of BVSMC that was treated with normal calcium phosphate concentration had little annexin II signal ( Fig. 7 B ). These data suggest that, in cultured BVSMC, high-calcium phosphate enhanced annexin II binding to fetuin-A at the cell membrane.
Fig. 7. Fetuin-A and annexin II bind on cell membrane in BVSMC. A : BVSMC were incubated in serum-free media with normal calcium phosphorus concentrations (normal Ca x P i ) or high calcium and phosphorus concentrations (high Ca x P i ) in the absence ( lanes 1 and 3 ) or presence of fetuin-A ( lanes 2 and 4 ), and cell membrane fractions of BVSMC were isolated. Western blot was performed to examine the quantity of annexin II in membrane. There was no difference in annexin II quantity in the cell membrane with different treatment. B : to analyze the interaction between annexins and fetuin-A, 500 µg of membrane protein were immunoprecipitated with antibody against fetuin-A. These fetuin-A containing membrane fractions were then analyzed by Western blotting using antibody against annexin II. The results demonstrate that high Ca x P i enhanced annexin II binding of fetuin-A at the cell membrane.
DISCUSSION
Fetuin-A is known to bind to calcium and phosphate in serum to decrease the supersaturation of serum and thereby reduce extraskeletal calcification. In addition, we and others have identified fetuin-A in arteries from chronic kidney disease and aging patients in areas of calcification ( 20, 21 ). As fetuin-A is not synthesized by VSMC, this implies that its deposition in areas of calcification may be a mechanism for local regulation of calcification. The present study demonstrates that VSMC can take up fetuin-A and that this uptake is dependent on calcium and is specific for fetuin-A (compared with dextran). Further supporting that this uptake may be a regulatory mechanism to control vascular calcification is our finding that the calcium-dependent cellular uptake of fetuin-A was also cell specific, only observed in VSMC and osteoblasts, the only cells tested that are capable of mineralizing. Fetuin-A constitutes a major component of the noncollagenous protein fractions of mineralized bone and plays an important role in bone formation and remodeling ( 1, 5 ). The fact that calcium-dependent fetuin-A uptake only occurs in BVSMC and osteoblasts, but not other types of cells, suggests that this pattern may be unique for mineralizing cells.
In addition to calcium, phosphorus is also a necessary and direct stimulus for calcification in BVSMC, with the effect of the two ions additive ( 21 ). However, we found no effect of phosphorus on fetuin-A uptake, consistent with work by Schinke et al. ( 25 ). They found 125 I-labeled fetuin-A coprecipitated in mixtures of calcium and phosphate and in mixtures of calcium and carbonate, but not in mixtures of magnesium and phosphate, indicating that the binding of fetuin-A to hydroxyapatite is due to interactions with calcium ions rather than with phosphate ions ( 25 ). Once endocytosed, the fetuin-A uptake led to sustained increases in intracellular calcium, indicating a secondary cell signaling process as a result of the uptake.
The mechanism of the calcium-dependent uptake of fetuin-A in our live cell imaging was fluid-phase as opposed to a receptor-mediated process, as the uptake was not saturable with unlabeled fetuin-A, yet dose dependent. We also demonstrated that the uptake was dependent on annexin activity, as we could block the cellular uptake with K201, a compound that has been shown to inhibit annexin calcium channel activity ( 11, 14 ). Furthermore, we found evidence for binding of fetuin-A and annexin II on cell membranes of BVSMC, and increased calcium and phosphorus significantly increase this interaction of fetuin-A and annexin II. The latter finding is consistent with a recent report of calcium-dependent binding of both annexin II and annexin VI to immobilized fetuin-A in a breast cancer cell line ( 17 ). These authors also found knockdown of annexin II or VI with small-interference RNA significantly reduced fetuin-A binding to the breast cancer cell surface ( 17 ). Thus annexin may serve as a "receptor" or "gate" for fetuin-A, but without saturable binding. It is more likely that fetuin-A or calcium or both alter annexins, leading to conformational changes at the cell membrane. Support for this are a number of studies demonstrating that annexins are multifunctional cell surface receptors that interact with a variety of extracellular ligands, such as plasminogen activator ( 9 ), heparin ( 12 ), and chondroitin sulfate chains ( 27 ).
We also demonstrated that fetuin-A induced a sustained increase in [Ca 2+ ] i in BVSMC. However, in the absence of extracellular calcium, there is a minimal increase in intracellular Ca 2+ induced by fetuin-A. This rise in intracellular calcium was completely blocked by inhibition of annexin activity, whereas the intracellular calcium rise induced by ionomycin was not. These data indicate that the source of increased intracellular Ca 2+ is from extracellular calcium. We hypothesize that fetuin-A, when it arrives at the membrane annexin "gate" with calcium, changes annexin conformation to allow both calcium and fetuin-A to enter the cells. If either fetuin-A is not carrying calcium, or annexins are blocked, then neither calcium nor fetuin-A enters the cells. This is plausible in that annexins are known to facilitate membrane conformational changes and the formation of lipid rafts that facilitate endocytosis in osteoblasts ( 7 ). Several studies have demonstrated that annexins are present on endosomal compartments and involved in endocytosis ( 6 ). For example, annexin II is found on early endosomes in complex with S100A10 to maintain the correct morphology of perinuclear recycling endosomes. Moreover, its depletion can interfere with the proper biogenesis of multivesicular endosomes from early endosomes ( 8 ). An alternative explanation is that annexins mediate calcium entry that then facilitates fetuin-A/annexin binding and fetuin-A endocytosis.
Our results strongly suggest annexin-mediated membrane organization and trafficking may be responsible for the fetuin-A uptake, which, in turn, may regulate the mineralization process in VSMC. Indeed, annexin has an important role in normal bone mineralization. Annexins II, V, and VI are highly expressed in hypertrophic and mineralizing growth plate cartilage, and the expression of annexins is indicative of terminal differentiation and chondrocyte mineralizing potential ( 15 ). In addition, annexins also form Ca 2+ channels in matrix vesicles, enabling Ca 2+ influx into these particles as an initial step for the formation of the first mineral phase in chondrocytes ( 14 ). Gillette and Nielsen-Preiss ( 7 ) overexpressed annexin II in osteoblasts, demonstrating a dramatic increase in alkaline phosphatase activity and mineralization. Our enhanced binding of annexin II and fetuin-A in BVSMC in conditions known to induce mineralization also support a role for annexin II in fetuin-A regulation of vascular calcification. Supporting this is recent data from Reynolds et al. ( 22 ), demonstrating that the presence of fetuin-A in matrix vesicles from VSMC renders them less able to mineralize. Clearly we are only beginning to understand this process, and the intracellular signaling pathways by which they may occur require further studies.
In conclusion, our study demonstrates that fetuin-A uptake by BVSMC is dependent on extracellular calcium and annexin activity, and that fetuin-A binds to annexin II at the cell membrane of BVSMC. Once endocytosed, fetuin-A induces a sustained rise in intracellular calcium, which may facilitate its regulatory role in VSMC calcification.
GRANTS
This work was supported by an unrestricted research grant from The Genzyme Corporation (N. X. Chen), K01 Grant from National Institute of Diabetes and Digestive and Kidney Diseases (N. X. Chen), and Veterans Affairs Merit Award (S. M. Moe).
DISCLOSURES
S. M. Moe has a consultancy with, and has received honoraria from the Genzyme Corporation.
ACKNOWLEDGMENTS
The authors thank Dr. Randall Duncan (University of Delaware) for providing technical advice regarding intracellular calcium imaging, and Michelle Murray for excellent secretarial assistance.
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作者单位：1 Indiana University School of Medicine, and 2 Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana