Localization of 1,25-(OH)2D3-responsive Alkaline Phosphatase in Osteoblast-like Cells (ROS 17/2.8, MG 63, and MC 3T3) and Growth Cartilage Cells in Culture*

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)zD3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is local- ized in extracellular matrix vesicles (MV). Since oste-oblast-like cells also have 1,25-(OH)zD3-responsive al- kaline phosphatase, this study determined whether the 1,25-(OH)zD3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco’s modified Eagle’s medium containing 10”-10-’2 M 1,25-(OH)zD3. Alkalinephosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity com- pared to PM (MV uersus PM: ROS 1 7 / 2 3 6 X; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose- dependent stimulation of MV alkaline phosphatase

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)zD3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)zD3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)zD3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10"-10-'2 M 1,25-(OH)zD3. Alkalinephosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV uersus PM: ROS 1 7 / 2 3 6 X; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dosedependent stimulation of MV alkaline phosphatase (5to 15-fold increase at iO"-10-~ M) in all cell types in response to the 1,25-(OHhD3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)zD3 was observed in growth cartilage PM. Although MV accounted for < 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)zD3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.
In contrast, alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is stimulated by 1,25-(OH)& These observations suggest that cells which calcify their matrix i n vivo produce alkaline phosphatase activity in cultures that is 1,25-(OH)zD3-responsive.
Recently, studies were reported (32, 33) which demonstrated that osteoblasts derived from fetal rat and mouse calvaria produce matrix vesicles in culture. When these cells were incubated with P-glycerophosphate, crystals were observed in association with the matrix vesicles. Since alkaline phosphatase and vitamin D3 have been implicated in hydroxyapatite formation, it was important to determine whether this enzyme is enriched in matrix vesicles produced by bone cells in culture and whether it is regulated by 1,25-(OH)& Accordingly, cells from two tissues, calcifying cartilage and bone, and three species (rat, mouse, and human) were examined.

EXPERIMENTAL PROCEDURES
Chondrocyte Cultures-Growth zone chondrocyte cultures were used as controls in this study since we had shown previously that they produced matrix vesicles in culture which contain 1,25-(OH)2D3responsive alkaline phosphatase activity (31). The system utilized has been described in detail by Boyan et al. (29). The costochondral growth zone cartilage was obtained by sharp dissection from the rib cages of 125-g Sprague-Dawley rats (30 rats/experiment), separated, sliced, and incubated overnight in Dulbecco's modified Eagle's medium (DMEM) with 5% COP in air at 37 'C. Chondrocytes were released from the cartilagenous matrix by sequential incubations in 1% trypsin (GIBCO) for 1 h and 0.02% collagenase (Worthington Type 11) for 3 h. Enzymes were prepared in Hanks' balanced salt solution. Digests were filtered (40 mesh nylon), and the cells were collected by centrifugation at 500 X g for 5 min, resuspended in  (34), glucocorticoids (35), and increased levels of bone alkaline phosphatase (36). The osteogenic nontransformed murine cell line, MC 3T3 was cloned from newborn mouse calvaria by Kodama et al. (37) and has been extremely well characterized. At subconfluency the cells have low levels of alkaline phosphatase; however, this level increases several hundred-fold when the cells reach confluency. This cell line has retained osteoblastic characteristics such as receptors for 1,25-(OH)J)3 (38) and parathyroid hormone (39), and it produces type I collagen (38), possesses bone alkaline phosphatase, and calcifies its matrix in culture (40). The cell line is, therefore, well suited for studies of 1,25-(OH)zD3 effects on osteoblasts. Although not as well characterized as ROS 17/2.8 or MC 3T3, the new human osteosarcoma cell line, MG 63, responds to 1,25-(OH)zD3 in a similar fashion to the two previously described cell lines by exhibiting an increase in alkaline phosphatase and inhibition of proliferation followed by morphological changes in response to 1,25-(OH)2DB (41). These three cell lines represent rat, mouse, and human tissues and were investigated in order to determine if the distribution of alkaline phosphatase and responsiveness to vitamin D3 metabolites, as seen in rat costochondral growth zone chondrocytes, is a generalized phenomenon of cells which mineralize their matrix.
For all bone cell cultures described below, the concentration of FBS in the media and the length of time for which the cells were incubated with hormone was determined in preliminary experiments as optimum for measuring alkaline phosphatase activity (42).
Confluent cultures of the rat osteosarcoma cell line, ROS 17/2.8, were plated in T-75 flasks or 96-well plates at 20,000 ce11s/cm2. Subconfluent cultures were plated at 10,700 cells/cm2. Both cultures were incubated in DMEM containing 10% FBS plus antibiotics for 24 h in 5% CO, at 37 "C. At that time, the media was removed and 1,25-(0H)*D3 added in DMEM containing 2% FBS. The plates were incubated an additional 48 h with hormone and then harvested as described for chondrocytes. The ROS 17/2.8 cells were a gift from Dr. Gideon Rodan, Merck Sharpe and Dohme.
The nontransformed murine cell line, MC 3T3, was plated in T-75 flasks or 96-well plates at 2000 cells/cm2 in DMEM containing 10% FBS. Cells were cultured for 48 h at which time the media was replaced with DMEM containing 5% FBS and the appropriate concentration of 1,25-(OH)&. Cells were cultured an additional 10 days and then harvested as above. MC 3T3 cells were a gift of Dr. M. Kumegawa, Josai University, Japan.
Human osteosarcoma MG 63 cells, obtained from the American Type Culture Collection, were plated at 9300 cells/cm2 in DMEM containing 10% FBS. After 24 h, the media was replaced with DMEM containing 2% FBS and the appropriate concentration of hormone. Cultures were harvested 4 days later.
Incubation with Hormones"l,25-(OH)2D3 was solubilized in ethanol immediately prior to use, and these stock preparations were diluted at least 1:5000 (v/v) with DMEM prior to addition to the cultures. Cultures with no additive and cultures incubated with ethanol at the same concentrations were used as internal controls. Ethanol concentration was ~0.0002%. lr25-(OH)2D3 was a gift of Dr. Milan Uskokovic of Hoffmann-LaRoche. Cell viability was determined by trypan blue dye exclusion at time of harvest.
Preparation of Membrane Fractions-Following incubation with hormone, chondrocyte and osteoblast cultures were washed twice with DMEM without FBS and trypsinized (1% in Hanks' balanced salt solution). Cells were collected, resuspended in saline, washed twice, and counted. Matrix vesicles were isolated from the digest supernatant by differential centrifugation, washed, and resuspended in 0.9% NaCl (29). Plasma membranes were prepared from cell homogenate5 (43) and resuspended in 1 ml of 0.9% NaC1. All samples used in subsequent assays represent the combination of three cultures (three T-75 flasks). Protein content of each fraction was determined (44).
Transmission Electron Microscopy-Matrix vesicle and plasma membrane pellets were fixed in phosphate-buffered 4% formaldehyde, 1% glutaraldehyde, post-fixed in 1% 0504, dehydrated, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Phillips 301 transmission electron microscope. Enzyme Activity-Specific activity of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase alkaline (EC 3.1.3.1)) was determined for both the cell layer and the isolated membrane fractions and measured as a function of p-nitrophenol hydrolysis from pnitrophenylphosphate at pH 10.2 (45).
Enzyme activity in the chondrocyte cell layer was measured following the method of Hale et al. (28). Confluent, second passage cells were subcultured to 24-well culture dishes (Coming) using a plating density of 25,000 cells/cm2. Hormone was added at confluency, and cultures were incubated an additional 24 h. At harvest the medium was decanted, and the cell layer was removed using a cell scraper. After centrifugation, the cell layer pellet was washed with phosphatebuffered saline and resuspended by vortexing in 500 p l of deionized water plus 25 pl of 1% Triton X-100.
Alkaline phosphatase activity in the osteoblast cell layer was measured using the assay described by Majeska et al. (24) scaled down to 96-well microtiter plates. The assay was performed as described previously (42). At harvest, the media was aspirated, the cells washed twice with phosphate-buffered saline, and 100 pl of 0.05% (v/ v) Triton X-100 was added to each well. The microtiter plate was freeze-thawed twice, and each well mixed with a Titre-tek pipettor. An additional 100 pl of 0.05% Triton was added only to the ROS 17/ 2.8 samples. Approximately 10-20% of the total cell lysate was used for the protein determination in all three cell lines. Approximately, 5-10% was used for the detection of alkaline phosphatase activity in the ROS 17/2.8 cells, whereas 50% was used for the MC 3T3 and MG 63 cell lines.
Specific activity of alkaline phosphatase was measured as follows. A protein standard consisting of 1-4 pg of human immunoglobulin G (Bio-Rad)/lGO pllwell was assayed in triplicate in a 96-well plate with Triton X-100 concentration taken into account. Bio-Rad protein reagent (40 pl/well) was added and each well immediately mixed with a Titre-tek pipettor. The plate was read at 600 nm on an EIA microtiter plate reader (M. A. Bioproducts) between 5 and 30 min after mixing. Alkaline phosphatase substrate and standard were made according to the protocol of Majeska et al. (24) with the following modifications. The standard, p-nitrophenol (Sigma) consisted of 2-14 pmo1/100 pl/well in AMP buffer (aminomethylpropanol, 0.5 M 2amino-2-methylpropanol, pH 10, 8 mM p-nitrophenylphosphate, 2 mM MgC12), in triplicate in a 96-well microtiter plate. 100 pl of 0.5 N NaOH was added to terminate the reaction after 10-15 min for the ROS 17/2.8 cells and after 60-90 min for the MC 3T3 and MG 63 cells and the plates read at 410 nm. Alkaline phosphatase specific activity was calculated as nanomoles of Pi/pg of protein/min. 5'-Nucleotidase (5'-ribonucleotide phosphohydrolase (EC 3.1.3.5)) was measured as ["C]adenosine released from [14C]AMP (46). This enzyme served as an internal reference of the relative purity of the membrane preparations. To facilitate comparison with the chondrocyte cultures, all data are expressed as micromoles of Pi/mg of protein/min. Statistical Analysis-All data are expressed as the mean * S.E. of six sample cultures or six wells. Figures and tables contain data from representative experiments. All experiments were performed a minimum of three times. Significance between data points and controls was determined using a two-tailed Student's t test using <0.05 confidence limits. Matrix Vesicle Purification-The matrix vesicle preparations used in this study appeared by transmission electron microscopy to be rich with matrix vesicles and relatively free of other cell and matrix components. Fig. 1 is an electron micrograph of the matrix vesicle pellet obtained from cultures of ROS 17/2.8 cells and is typical of all of the matrix vesicles isolated from cartilage and bone cell cultures in this study. The vesicles were 0.05-0.2 pm in diameter and contained granular amorphous material. No crystals were present in the matrix vesicles. Fig. 2 is an electron micrograph of the plasma membrane pellet obtained from cultures of ROS 17/2.8 cells and is typical of all the plasma membrane preparations isolated in this study. The membranes were free of mitochondria, nuclear material, and matrix vesicles.

Cell
Effect of 1,25-(0f&D3 on Cell Number-Bone cell number was decreased by 1,25-(OH)zD3 (Table I). The effect of hormone was greatest in the MC 3T3 cultures and least in the ROS 17/2.8 cultures. The decrease was statistically significant in MG 63 cells and MC 3T3 cells at 10-7-10-9 M but was observed in ROS 17/2.8 cells at M only. There was a similar effect of hormone on subconfluent cultures as well (data not shown). The decrease in cell number observed was not due to a toxic effect of hormone. The viability of cells treated with or lo-' M 1,25-(OH)zD3, as determined by trypan blue dye exclusion, was identical to, or not significantly different from, nontreated controls for all cell lines examined. were harvested by trypsin digestion of the extracellular matrix. Matrix vesicles were isolated from the trypsin digests following pelleting of the cells and differential centrifugation. Pellets were fixed in buffered glutaraldehyde and post-fixed in Os04 and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined on a Phillips 301 electron microscope. Bar represents 1 pm; arrows indicate matrix vesicles.

FIG. 2. Transmission electron micrograph of the plasma membrane fraction of confluent cultures of ROS 17/23 cells.
Plasma membranes were isolated by differential centrifugation, fixed in buffered glutaraldehyde, post-fixed in Os04, and embedded in Epon. Bar represents 0.5 pm.  Because of the decrease in cell number, all analyses of enzymatic activity were calculated both with respect to cell number and as a function of the protein content of the specific fraction.
Distribution of Alkuline Phosphatase-Distribution of alkaline phosphatase activity in cultures of growth zone chondrocytes confirmed that reported previously (31). ROS 17/2.8 cells exhibited enrichment of alkaline phosphatase specific activity in the matrix vesicle pellet when compared to the cell layer (86-fold) or to the plasma membrane (6-fold) ( Table 11). The distribution of activity in the ROS 17/2.8 cells was similar to that seen in the mouse and human cell lines (data not shown). Although the percent recovery of total activity in the matrix vesicle and plasma membrane fractions was comparable to that of growth zone chondrocytes (31), specific activity was higher in the chondrocyte cultures and specific membrane fractions (cell layer, 24 x; matrix vesicles, 3 x; plasma membranes, 9 X).
Enrichment of specific activity of alkaline phosphatase in the matrix vesicles with respect to the cell layer was markedly greater in the bone cell cultures than in the chondrocytes (chondrocytes, 11 X; ROS 17/2.8,86 X). Similar enrichment of alkaline phosphatase activity in the matrix vesicles produced by MC 3T3 cells (Table 111) and MG 63 cells (Table  IV) was observed, when compared to the cell layer (Table V).   The specific activity of 5'-nucleotidase was also enriched in the matrix vesicles with respect to the plasma membranes in all osteoblast cell lines examined (Table VI). There was a 6.4-fold enrichment of this enzyme activity in matrix vesicles produced by MG 63 cells, a 4.3-fold enrichment in matrix vesicles produced by ROS 17/2.8 cells, and a 2.2-fold enrichment in matrix vesicles produced by MC 3T3 cells. Enzyme activity in the plasma membranes of all three osteoblast cell lines was essentially the same, averaging 148 cpm/mg protein/ min.    M with peak stimulation occurring at M (Fig. 3). Although stimulation of enzyme activity in the subconfluent cultures was observed at the same concentrations of hormone, peak stimulation was at lop6 M 1,25-(OH)*D3 (Fig. 4). In addition, the magnitude of response differed with respect to the degree of confluency at the time hormone was added to the cultures. Basal enzyme specific activity was two to three times higher in the confluent cultures. The subconfluent cultures incubated with 1,25-(OH)2D3 exhibited alkaline phosphatase activity comparable to that of the confluent control cultures. In contrast, alkaline phosphatase specific activity in the confluent cultures incubated with lo-? M 1,25-(OH)zD3 was increased approximately 400%.

Distribution of 5'-nwleotidase activity in the plasma membranes and matrix vesicles isolated from confluent cultures of ROS 1712.8, MC 3T3, and MG 63 osteoblast-like cells
Although specific activity of alkaline phosphatase was significantly lower in the MG 63 and MC 3T3 cultures, both cell types showed a dose-dependent increase in response to hormone. The confluent MG 63 cells exhibited a statistically significant 2.5-fold increase in enzyme activity at io-' M 1,25-(OH)zD3. At lo-' M, the increase was 12-fold. The increase in MC 3T3 alkaline phosphatase was significant at M and was elevated 2.5-fold at M 1,25-(OH)2D3. As was observed with ROS 17/28 cells, the basal activity of subconfluent cultures was lower, and at lO"-lO-' M 1,25-(OH)zD3, it approached that of the basal levels of the confluent cell layer (data not shown).
Effect of 1,25-(oH)zD3 on Plasma Membrane and Matrix Vesicle Alkaline Phosphatase Actiuity-Stimulation of total specific activity in the cell layer of all bone cells was observed at significantly lower sensitivities than were possible by measuring the isolated membrane fractions. Matrix vesicles isolated from confluent ROS 17/2.8 cultures exhibited a dosedependent increase in enzyme activity which was statistically significant at 10-s-10-7 M 1,25-(OH)2D3 (Fig. 5). The magnitude of response was independent of denominator, i.e. cell number or matrix vesicle protein content. Similarly, matrix vesicles from subconfluent cells exhibited a similar increase in specific activity, significant at lo-' M (Fig. 6). The absolute levels of basal activity were comparable to those of matrix vesicles isolated from confluent cultures with respect to cell number, but the magnitude of stimulation was slightly lower in the subconfluent cells at comparable hormone concentrations. Peak stimulation in confluent cultures was observed at  teaued in the subconfluent cultures at M. When enzyme activity was calculated with respect to matrix vesicle protein content, basal levels in the subconfluent cultures were approximately one-half that of the confluent cultures. The -fold stimulation was comparable, but peak levels were three times greater in matrix vesicles isolated from the confluent cells.
Basal levels of alkaline phosphatase in MC 3T3 cells were 0.8 +. 0.5 pmol of Pi/mg of protein/min (Table V) However, the basal level in the plasma membranes of these same cells was 0.9 & 0.1 pmol of Pi/mg protein/min and at M 1,25-(OH)zD3 was 2.0 f 0.2 (Table 111). The sensitivity afforded by measuring activity in the matrix vesicles was 33fold greater than the plasma membranes and 37-fold greater than the cell layer. This sensitivity was evident in control cultures and in cultures incubated with 1,25-(OH)zD3. Enhancement of sensitivity was observed in matrix vesicles produced by MG 63 cells, although the magnitude of enhancement was not as great (Table IV).
In contrast to chondrocytes, 1,25-(OH)zD3 stimulated both the plasma membrane and matrix vesicle enzymes in bone cells. The magnitude of stimulation depended on the bone cell type and on the denominator. For instance, stimulation of matrix vesicle alkaline phosphatase was observed at lo-' M 1,25-(OH)zD3 was 12.5 X in MC 3T3 cultures (Table III), 3.5 X in MG 63 cells (Table IV), and 2.2 X in ROS 17/2.8 cultures (Fig. 5), when data was calculated with respect to the cell number. In contrast, no differences in stimulation were detected when data were calculated as a function of matrix vesicle protein (MC 3T3, 2.1 x; MG 63, 2.0 x; ROS 17/28, 1.9 X). Stimulation of the plasma membrane enzyme at IO-' M 1,25-(OH)2D3 was essentially the same for all three cell types and independent of denominator except for ROS 17/28 cells (micrograms of Pi/106 cells/min: MC 3T3,3.3 X; MG 63, 4.4 X; ROS 17/2.8, 9.5 x uersus micrograms of Pi/mg of protein/min: MC 3T3, 2.0 X; MG 63, 3.2 X; ROS 17/23, 2.3 x).

DISCUSSION
Primary mineralization, characterized by the occurrence of extracellular matrix vesicles, has been described in various normal and pathologic tissues, including developing epiphyseal cartilage, embryonic bone, bone wound repair, mantle dentin, ossifying neoplasms, and ectopic calcifications (9,(47)(48)(49)(50)(51)(52). In calcifying tissues like growth cartilage and bone, mineralization is regulated by vitamin D and is associated with increased alkaline phosphatase activity, an enzyme that is enriched in the matrix vesicles isolated from cartilage. Although other papers have reported the influence of 1,25-(OH),D3 on alkaline phosphatase in cell layers (24,28,42), there has been no effort to distinguish or pinpoint the actual location of the stimulated enzyme activity beyond release into culture media. Therefore, any effect of the vitamin D3 metabolite was ascribed to the cell population as a general phenomenon.
The current investigation has shown that the response to 1,25-(OH)2D3 is site-specific; the matrix vesicles are targeted and not the cell population per se. In the membrane and matrix vesicle preparations, half of total specific activity and most of the 1,25-(OH)2D3-responsive enzyme specific activity in osteoblast cultures is localized to the matrix vesicles. Furthermore, other investigators have utilized methodologies that separated the cell from the matrix prior to measurement of enzyme activity. Thus, only the cell population was examined for alkaline phosphatase activity, the matrix being discarded (25)(26)(27), therefore disregarding the significance of the alkaline phosphatase activity in the matrix and its regulation. This is no small oversight since recently matrix vesicles have been shown to mediate calcification by osteoblasts in vitro (32).
The matrix vesicles isolated in this study did not contain any evidence of crystals. Whether this was due to their loss during sample preparation or that they were not present in the cultures originally could not be determined under the experimental parameters used. Similar results have been obtained by other laboratories (14) as well as our own (29) using these procedures to isolate matrix vesicles from rat chondrocyte cultures. The matrix vesicles were also morphologically comparable to matrix vesicles observed in cartilage (9) and bone (53) in situ.
The data presented in this study support the theory that matrix vesicles are distinct organelles, although they are derived from the plasma membrane (54). For all osteoblast cell lines examined, the plasma membrane marker enzymes, alkaline phosphatase and 5'-nucleotidase, exhibited enriched activity in the matrix vesicle fraction. Previous observations in our laboratory (29) have shown that matrix vesicles produced by resting zone cells are also enriched in 5"nucleotidase. Those produced by growth zone chondrocytes have elevated levels of this enzyme, but it is not statistically greater than that of the plasma membrane. Thus, specific enrichment of this enzyme does not appear to be a general characteristic of cells which calcify their matrix. Activity in the plasma membranes is comparable for all osteoblasts and is in the same order of magnitude as that of the growth zone chondrocytes (29). However, the fact that there is considerably more enrichment in the matrix vesicles produced by osteoblasts demonstrates fundamental biochemical differences in this organelle as it is produced by chondrocytes and osteoblasts. This observation is supported by the fact that the alkaline phophatase: 5'-nucleotidase ratio varies between chondrocyte and osteoblast and among osteoblast cell lines.
The data presented in this paper indicate that the membrane isolation technique used yields those fractions of the culture which are most highly enriched in alkaline phosphatase specific activity, suggesting that enzyme present in the plasma membrane and matrix vesicles will be most responsive to metabolic regulation of the mineralization process. Since these two fractions represent only 34% of the total enzymatic activity, it is probable that subtle changes in alkaline phosphatase regulation might be missed if the entire culture were assayed. This is especially true for matrix vesicles which represent less than 20% of the total activity, yet are the sites most intimately involved in the mineralization process and the most highly enriched fraction in terms of specific activity. Because 1,2D3 had an inhibitory effect on cell number, it was important to separate the influence of cell number from the influence of the amount of protein in a specific fraction in understanding the mechanism of hormone regulation. This is an important distinction. As demonstrated in Table 111, the effect of 1,25-(OH)2D3 on matrix vesicles and plasma membranes isolated from MC 3T3 cells appears to be much greater when the data are expressed as a function of cell number rather than as specific activity of the isolated membrane fraction. Since the stimulation in the matrix vesicle specific activity is only 2-fold, the data may suggest that fewer cells are producing more vesicles per cell. Alternatively, the vesicles themselves may contain more total protein of which the stimulation in alkaline phosphatase is only 2-fold. Similar results were observed in cultures of MG 63 cells (Table   IV) and in cultures of ROS 17/22 cells (Figs. 5 and 6). It is unlikely that the stimulation of alkaline phosphatase is merely a mathematical consequence of reduced cell number due to vitamin D toxicity, since the trypan blue dye exclusion studies demonstrated that the cells remained viable. Even though the cells were at confluence when hormone was added, they did continue to divide. This is a property of the chondrocytes and osteoblasts in cultures which we have noted routinely.
It was not surprising that matrix vesicles and plasma membranes isolated from the chondrocyte cultures respond so differently to 1,25-(OH),D3. Although matrix vesicles are derived from the plasma membrane, they have a distinctly different biochemical composition. Both phospholipid composition (17,29) and enzymatic activities (7,30,31) differ from those of the parent membrane. In fact, alkaline phosphatase is routinely enriched 2-to 3-fold in even crude matrix vesicle preparations (7) and, as a result, serves as the matrix vesicle marker enzyme. These observations suggest that matrix vesicle biogenesis and/or maturation yields a membrane capable of responding in a unique fashion to hormone signals. Since at least one function of matrix vesicles is to promote initial hydroxyapatite deposition (9), it may be that the action of vitamin D on these membranes in particular is to facilitate that process. This appears to be the case for bone cells as well. Difference in the behavior of the matrix vesicles isolated from the two cell types may be due to differences in their composition or in their regulation by the cell itself.
The results support the hypothesis that 1,25-(OH)& may serve as a regulating influence on alkaline phosphatase specific to the matrix vesicle fraction (55). It is not known whether this regulatory effect is on the matrix vesicle in the matrix or on the cell membrane prior to the formation of the matrix vesicle. When data are normalized to cell number, the effect of hormone in bone cells is comparable in both fractions, suggesting that at least one regulatory mechanism is by incorporation of new enzyme into the plasma membrane and subsequent release into the matrix as matrix vesicles, probably through activation of alkaline phosphatase gene transcription (56). The second level of regulation then occurs as a function of the differences in protein content (and composition) of the matrix vesicles and plasma membranes, resulting in a significant increase in specific activity in the matrix where mineralization is occurring. Although the rate of stimulation is the same in both membrane populations, the effect in the matrix is more activity.
The elevation of chondrocyte alkaline phosphatase activity by 1,25-(OH)zD3 observed in this study was parallel to that reported for cartilage slices in culture (57). Similarly, the stimulation of the osteoblast was comparable to that reported by other laboratories (23,24,34,38,41,58). Recently reported decreases in alkaline phosphatase activity in response to 1,25-(OH)& in chondrocytes grown in serum-free media (28) might have been due to the response of these cells to an initial exposure to hormone. Initial exposure to hormone might also account for the reported physiologic concentration of vitamin DB at 1 0 " ' M. In the current study, chondrocyte and osteoblast cultures were acclimated to basal levels of hormone in the FBS-containing media; therefore, alkaline phosphatase activity was measured in response to additional hormone, and this may account for our observed physiologic concentration of hormone to be greatest at lo-' and M in confluent cells and M in subconfluent cells. Some of the differences in the magnitude of response noted for the chondrocytes and each of the osteoblast cell lines may have been a function of the differences in serum used in the media, resulting in varying endogenous hormone levels and, as a result, varying basal enzyme activity.
The degree of confluence is also a factor in interpreting the response of bone cells to hormone. Although stimulation of hormone is observed in both types of cultures, the magnitude is markedly different. The fact that subconfluent cultures incubated with hormone approximate basal levels of confluent cells may suggest that 1,25-(OH)& is promoting cell differentiation at the expense of cell proliferation. This interpretation is supported by the observation that basal activities in matrix vesicles isolated from both cell cultures are comparable, especially when normalized to cell number. Differences in specific activity may be due to additional maturation of the matrix vesicles which occurs in confluent cultures only or because more mature vesicles are no longer being diluted by newly synthesized matrix vesicles in the subconfluent cultures. Thymidine incorporation studies (30) have demonstrated that cell proliferation is still occurring in the confluent cultures, although at much reduced rates, and this is further suppressed by exogenous hormone.
The subtle differences in osteoblast response would have been missed if stimulation of enzyme activity in the cell layer had been the sole parameter. For instance, in cultures of MC 3T3 cells, incubated with lo-' M 1,25-(OH)2Da, only a 25% increase in alkaline phosphatase specific activity was seen.
There was a 60% increase in ROS 17/2.8 cells. Only in the MG 63 cells was a significant increase, 500%, observed at that hormone concentration. For all of the cell types, specific activity in the cell layer could be accounted for by that of the plasma membrane alone, thus obscuring the significant contribution of the matrix vesicle enzyme.
The data, therefore, indicate that the vitamin DS regulation of alkaline phosphatase at specific sites and in specific membrane populations, as first shown in rat costochondral growth zone chondrocytes, may be a generalized phenomenon unique to calcifying tissues. These results support the concept that not only in cartilage, but also in bone, primary mineralization is mediated by vitamin D3 responsive matrix vesicles. These data are all the more significant in light of recent publications demonstrating that osteoblasts may utilize matrix vesicles as mediators of mineralization in vitro (32,331. Extensive morphometric (59) and biochemical (60) studies using ablation of bone marrow to induce bone repair indicate that primary bone formation in vivo may be matrix vesicle-mediated as well.