tissue-nonspecific alkaline phosphatase is required for the calcification of collagen in serum

30 Pages·2008·9.86 MB·English

JBC Papers in Press. Published on December 19, 2008 as Manuscript M803205200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M803205200
TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE IS REQUIRED FOR THE
CALCIFICATION OF COLLAGEN IN SERUM: A POSSIBLE MECHANISM FOR
BIOMINERALIZATION.

Paul A. Price*, Damon Toroian, and Wai Si Chan

From the Division of Biological Sciences, University of California, San Diego, La Jolla, Ca.
92093-0368

Running title: Alkaline phosphatase is required for collagen calcification in serum

Address correspondence to: Dr. Paul A. Price, Division of Biological Sciences, 0368, University
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of California, San Diego, La Jolla, Ca 92093-0368. Phone: [phone]; Fax: [phone]; ow
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email: [email protected] lo
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ed
Previous studies have shown that the type serum nucleator of apatite crystal from
I collagen of tendon and demineralized formation. http
bone both calcify rapidly in serum. The Based on these and earlier studies, ://w
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speed, collagen-matrix-type specificity, the mechanism of collagen calcification in w
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and extent of the re-calcification of serum requires at least four elements: 1. c
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demineralized bone in serum suggest that A matrix (collagen fibrils) that is brg/
the serum calcification activity identified accessible to small apatite crystals but not y g
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in these studies may participate in normal large molecules (JBC 282: 22437-47). 2. es
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biomineralization. A large serum nucleator that generates n J
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Because of its presence in serum small crystals, some of which diffuse into nu
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and its long history of association with the the fibrils. 3. A source of TNAP to ry 2
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normal mineralization of the collagen activate the serum nucleator. 4. A large , 2
0
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matrix of bone, tissue-nonspecific alkaline protein (fetuin) that selectively inhibits 9
phosphatase (TNAP) is an obvious growth of crystals remaining in solution,
candidate for a protein that could be a thereby ensuring that only crystals within
component of serum calcification activity, fibrils grow (CTI 82: 116-126).
and experiments were therefore carried
out to test this possibility. These Our goal is to understand the
experiments show that the inactivation of biochemical mechanism responsible for the
TNAP in serum prevents collagen calcification of collagen fibrils in normal
calcification, and that the addition of bone formation. In the course of our
physiological levels of purified TNAP investigations, we have discovered that
restores the ability of TNAP-deficient purified type I collagen and demineralized
serum to calcify collagen. Additional bone matrix both calcify rapidly when
experiments show that the role of TNAP incubated in serum in the absence of cells
in collagen calcification is to activate a (1-4). The calcification of collagen is due to
the presence of a serum calcification
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
2
activity, one sufficiently potent that collagen We have recently published two studies
calcifies when incubated in media on the mechanism of serum-induced
containing as little as 1.5% serum but not in calcification of collagen fibrils (6); (7). The
serum-free media alone (1-4). This serum collagen fibril is about 70% water by
calcification activity consists of one or more volume (8), and during mineralization the
proteins that are 50 to 150 kDa in size (3,4). water within the fibril is replaced by mineral
Although serum-driven collagen (9-11). Our first study demonstrated that the
calcification is an in vitro, cell-free assay, water within the fibril is accessible to
there are several reasons to believe that it molecules and apatite crystals as large as a 6
could be relevant to understanding kDa protein, and inaccessible to molecules
mechanisms by which collagen fibrils are larger than a 40 kDa protein (7). Our second
mineralized in nature. 1. The assay study showed that fetuin, a 48 kDa serum
conditions are physiologically relevant: inhibitor of mineral growth (12,13),
collagen added to serum calcifies when determines the location in which the apatite
incubated at the temperature and pH of crystals generated by the serum calcification
mammalian blood, without the need to add activity can grow: in the absence of fetuin
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anything to serum to promote mineral grows in the solution outside ow
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mineralization, such as β glycerophosphate collagen fibrils, while in the presence of loa
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or phosphate (see (1) and references fetuin mineral grows exclusively within ed
therein). 2. Serum is relevant to bone collagen fibrils (6). from
mineralization: osteoblasts form bone in a The results of these studies show that the http
vascular compartment (5), and proteins in mechanism of serum-induced calcification ://w
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serum have direct access to the site of requires at least three elements: 1. A matrix w
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collagen fibril formation and mineralization with an interior aqueous compartment that is c
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while proteins secreted by the osteoblast accessible to small apatite crystals but not brg/
appear rapidly in serum. 3. Serum-driven large molecules. 2. A large nucleator (not y g
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calcification is evolutionarily conserved: the yet identified) that generates small crystals es
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serum calcification activity appeared in outside of the matrix, some of which diffuse n J
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animals at the time vertebrates acquired the into the matrix. 3. A large protein (fetuin) nu
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ability to form calcium phosphate mineral that selectively inhibits the growth of those ry 2
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structures, with no evidence for a similar crystals remaining in solution outside the , 2
0
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activity in the serum of invertebrates (2). 4. matrix, thereby ensuring that only crystals 9
Serum-driven calcification is specific: within the matrix grow. We have proposed
calcification is restricted to those structures the term ‘Shotgun Mineralization’ for this
that were calcified in bone prior to calcification mechanism (6): Crystals form
demineralization, with no evidence of throughout the solution, and only those that
calcification in cartilage at the bone ends or diffuse into a matrix grow.
in cell debris (1,3). 5. Serum-driven Among the known serum proteins,
calcification can achieve the total re- tissue-nonspecific alkaline phosphatase
calcification of demineralized bone: serum- (TNAP1) is arguably the most obvious
driven calcification progresses until the re- candidate for a protein that could be a
calcified bone is comparable to the original component of serum calcification activity.
bone in mineral content and composition, TNAP has a long and extensive history of
radiographic density, and powder X-ray association with normal bone calcification
diffraction spectrum (3). (14), and the molecular weights of the
different glycosylated forms of the serum
3
enzyme range from 126 to 141 kDa (15), mm (60 mesh) sieve, demineralized by
which is within the molecular weight range extraction for 72h with 0.5 M EDTA pH 7.5,
expected for serum calcification activity (3). and then washed exhaustively with water
Serum TNAP consists of approximately and dried. Tibias were dissected from
equal amounts of the bone and liver newborn rats, demineralized in 0.5 M EDTA
isoforms of the enzyme. These isoforms pH 7.5 for 72h at room temperature using a
have the same protein sequence but differ in 300 fold molar excess of EDTA to mineral
O-linked glycosylation (22). Tissue- calcium, washed exhaustively with ultra
nonspecific alkaline phosphatase is a zinc pure water to remove all traces of EDTA,
metalloenzyme that hydrolyzes a broad and stored at –20ºC until use (3). Rat tail
range of phosphate monoesters, but its tendons were prepared as described (3).
normal physiological substrate is presently Bovine kidney alkaline phosphatase (tissue-
unclear (14). As its name implies, tissue- nonspecific alkaline phosphatase, TNAP)
nonspecific alkaline phosphatase is with a specific activity of 1000 U/mg was
expressed in a wide variety of tissues, obtained from Calzyme Laboratories (San
tissues that include bone, liver, and kidney Luis Obispo, CA); Beryllium sulfate
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(14). In spite of its nonspecific expression tetrahydrate from Fluka; and (-)-tetramisole ow
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in tissues, however, genetic deficiency in hydrochloride (levamisole), 1,10- lo
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tissue-nonspecific alkaline phosphatase in phenanthroline monohydrate, purified ed
mice and humans (called hypophosphatasia) bovine milk casein, purified type I collagen from
is primarily associated with defects in bone from bovine achilles tendon, and Alizarin http
mineralization (14,16,17). This observation, red S from Sigma. Newborn calf serum was ://w
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together with abundant expression of purchased from Invitrogen Corp. (Carlsbad, w
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alkaline phosphatase at sites of bone CA), and had a basal alkaline phosphatase c
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mineralization (18,19) in association with activity of 0.2 U/ml. A single batch of brg/
the outer surface of osteoblasts (20) and newborn calf serum was used for all y g
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matrix vesicles (21) supports a probable role experiments in order to eliminate serum as a es
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of the enzyme in normal bone possible variable. DMEM was prepared by n J
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mineralization. supplementing each 500 ml volume of nu
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The present experiments were Dulbecco's modified Eagle medium (Gibco) ry 2
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carried out to determine whether the with 5 ml of penicillin-streptomycin , 2
0
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endogenous tissue-nonspecific alkaline (Gibco), 1 ml of 10% sodium azide, and 1.1 9
phosphatase found in serum is a component ml of 0.5 M sodium phosphate buffer pH 7.4
of serum calcification activity. The results (final medium P concentration = 2 mM).
i
of these experiments show that TNAP is an Experiments with neat serum. In a typical
essential component of serum calcification re-calcification experiment, 4 mg
activity, and that its role in the Shotgun demineralized bovine bone sand (dry
Mechanism is to activate the serum weight) was re-hydrated in water, placed in
nucleator of apatite crystal formation. 2 ml newborn calf serum in a 24 well plate
(Costar 3524), and incubated in a humidified
EXPERIMENTAL PROCEDURES incubator at 37ºC and 7.5% CO with
2
Materials. Bovine bone sand was prepared continuous, gentle mixing using a plate
from the midshaft of bovine cortical bone as shaker. In the calcification time course
described (23). The bone sand was sized to experiment (Figure 1), 3 wells contained 4
obtain particles that pass through a 0.43 mm mg bone sand in 2 ml new born calf serum
(35 mesh) sieve and are retained by a 0.25 (NBCS); 3 contained bone sand in NBCS
4
plus 10 U/ml TNAP; 3 contained bone sand incubator at 37ºC and 7.5% CO . The serum
2
in NBCS plus 30 mM levamisole; 3 was then placed into dialysis tubing
contained bone sand in NBCS plus 30 µM (MWCO, 6-8 kDa) and dialyzed against four
Beryllium sulfate; and 3 contained NBCS changes of 125 ml DMEM each using the
alone. At t = 0 and every 12 h thereafter, 25 same incubator. The same dialysis
µl were removed from every well, combined procedure was also used to remove any non-
with 25 µl of 150 mM HCl, and analyzed for protein associated zinc from the TNAP
calcium; calcium uptake was determined solution. Control serum was prepared by the
from the decrease in serum calcium due to same procedure using methanol vehicle
the presence of bone sand. To test the effect alone. TNAP activity-deficient and control
of added TNAP on calcification of different serum samples were frozen until use.
matrices (Figure A), rat tail tendon Experiments to examine the effect of
segments and single demineralized newborn TNAP-depletion on the calcification of
rat tibias were each separately incubated for collagen matrices were carried out using 24-
36 h in 2 ml volumes of NBCS and of well cell culture clusters (Costar 3524,
NBCS containing 10 U/ml TNAP. To Corning) in a humidified incubator at 37ºC
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determine the effect of alkaline phosphatase and 5% CO2. Each well contained 2 ml of own
inhibitors on bone re-calcification in serum, DMEM with 2mM Pi and 1% by volume of lo
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4 mg demineralized bone sand was one of the following: control newborn calf ed
incubated for 72 h in 2 ml NBCS containing serum; alkaline phosphatase-depleted serum; from
the indicated concentrations of beryllium or alkaline phosphatase-depleted serum + http
(Figure B), levamisole (Figure C), and 0.2 U/ml of purified TNAP (see Table 3). ://w
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1,10-phenanthroline (Figure 2). The amount of matrix added to each 2 ml w
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To separately assess the effects of volume was: a single hydrated, c
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alkaline phosphatase inhibitors on the demineralized newborn rat tibia; or a portion brg/
initiation and growth of calcification in of purified type I collagen from bovine y g
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demineralized bone (Figure 3), a 24 h pre- Achilles tendon (3 mg dry weight; hydrated es
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incubation in serum was used to introduce before use). Each tissue was then incubated n J
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mineral foci within the collagenous matrix, for 6 days. nu
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and a subsequent incubation for 5 d in Experiments with fetuin-deficient serum ry 2
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DMEM without serum was used to foster in DMEM. Fetuin-depleted serum was , 2
0
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growth of the initial crystal nuclei, using prepared as previously described (6). In 9
procedures that have been described brief, a column packed with rabbit anti-
elsewhere (3). Alkaline phosphatase bovine fetuin antibodies conjugated to
inhibitors were added at the serum initiation Sepharose 4B was equilibrated with DMEM,
stage or at the DMEM growth stage. and adult bovine serum was dialyzed against
Experiments with TNAP-deficient serum the same solution. The dialyzed bovine
in DMEM. Serum deficient in alkaline serum was then freed of fetuin by passing a
phosphatase activity was prepared using 0.85 ml serum aliquot over the column at
1,10 phenanthroline, a zinc chelator that room temperature. The absorbance at 280
irreversibly inactivates the enzyme (24). In nm of each 1 ml fraction was determined;
a typical experiment, 150µl of 1M the 4 fractions with the highest absorbance
phenanthroline in methanol was combined were then pooled and diluted with DMEM
with 10 ml of newborn calf serum in a 15ml until the absorbance equaled that of 10%
conical tube, and the tube was loosely bovine serum. Protein bound to the column
capped and placed for 72h in a humidified
5
was removed by washing the column with matrix surface to volume and thereby
100 mM glycine pH 2.5. enhance the diffusion of calcium, phosphate,
The effect of alkaline phosphatase or small crystals into collagen. In the 2 ml
and its inhibitors on apatite crystal formation serum volume used for this test there is only
in DMEM containing 10% fetuin-depleted sufficient calcium and phosphate to restore
serum was carried out using 24-well cell approximately 5% of the mineral that was
culture clusters in a humidified incubator at present in the bone sand prior to
37ºC and 5% CO (see (6)). demineralization.
2
Biochemical analyses. The procedures Figure 1 shows that the addition of
used for Alizarin red and von Kossa staining purified bovine TNAP to serum markedly
have been described (1). For quantitative accelerates the rate at which serum calcium
assessment of mineral formation, matrices is taken up by bone in this experimental
and precipitates were extracted for 24 h at system, with half maximal calcium uptake at
room temperature with 1 ml of 0.15 M HCl. 38 h in serum plus TNAP compared to 60 h
Calcium levels were determined in serum alone. The results of a repeat
colorimetrically using cresolphthalein experiment were similar, with half maximal
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complexone (JAS Diagnostics, Inc., Miami) calcium uptake at 40 h in serum plus added ow
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and phosphate levels were determined as TNAP compared to 64 h in serum alone (not lo
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described (25). Alkaline phosphatase shown). Although the addition of TNAP ed
activity was measured at pH 10.4 using p- substantially accelerates the re-calcification from
nitrophenol phosphate as substrate (JAS of bone in serum, it does not significantly http
Diagnostics). affect the amount of calcium taken up by ://w
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bone after 6 days incubation (Figure 1) or w
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RESULTS the total calcium and phosphate recovered c
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Evidence that the addition of from the re-calcified bone (Table 1). This brg/
alkaline phosphatase accelerates the rate result may reflect the effects of calcium and y g
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of collagen calcification in serum. Tissue phosphate depletion during the 6 day es
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nonspecific alkaline phosphatase (TNAP) is incubation on the final extent of mineral n J
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abundantly expressed at sites of bone formation in collagen, since most of the nu
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mineralization (18,19), where it is held in calcium and phosphate initially in the 2 ml ry 2
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the external leaflet of osteoblast plasma serum aliquot has been deposited in bone by , 2
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1
membrane by a hydrophobic the 6 day time point. Previous studies have 9
glycosylphosphatidylinositol (GPI) anchor shown that the initial step in serum-induced
(32), and the local concentration of the apatite formation is highly dependent on the
enzyme near the osteoblast plasma concentration of calcium and phosphate in
membrane is likely to be far higher than the solution, and will not continue to occur
0.2 U/ml of alkaline phosphatase activity when the calcium phosphate ion product
found in calf serum. As a first step in falls below about 2.5 mM2 (1,2) (personal
evaluating the effect of high local observations).
concentrations of TNAP on serum-induced Figure 1 also shows that the addition
collagen calcification, we investigated the to serum of the alkaline phosphatase
impact of adding 10 U/ml of purified TNAP inhibitors beryllium (a competitive inhibitor
on the rate of bone re-calcification in new (26)) and levamisole (an uncompetitive
born calf serum at 37°C and pH 7.4. inhibitor (27,28)) completely prevented the
Demineralized bovine bone sand was used uptake of serum calcium by bone, and Table
for this test in order to increase the ratio of 1 shows that bone samples incubated in
6
serum with either beryllium or levamisole demonstrate that alkaline phosphatase
had no detectable calcium or phosphate, and addition to serum has a comparable ability
did not stain with Alizarin red. to accelerate the calcification of type I
A potentially trivial explanation for collagen matrices that were once calcified
the effect of alkaline phosphatase on the (demineralized tibias or bone sand) as well
kinetics of bone re-calcification in serum is as collagenous matrices that were not
that the enzyme could liberate sufficient previously mineralized (tail tendon).
phosphate from serum substrates to increase Dose dependent effects of alkaline
total ionic phosphate levels in serum, and phosphatase inhibitors on the
that this increase could accelerate the re- calcification of collagen in serum. The
calcification of demineralized bone. To test next experiments examined the dose
this possibility, serum containing the 10 dependent effects of three different alkaline
U/ml level of added TNAP was incubated phosphate inhibitors on the calcification of
for 9 days at 37°C in the absence of bone collagen in serum. Figure 2 shows that the
matrix. As can be seen in Table 2, serum re-calcification of demineralized bone in
phosphate levels were not increased by the serum is dose-dependently inhibited by
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9-day incubation with TNAP, and were also 1,10-phenanthroline, a zinc chelator that ow
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not affected by the addition of alkaline irreversibly inactivates alkaline phosphatase lo
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phosphatase inhibitors. This experiment (24), with no evidence of Alizarin red ed
shows that any substrate hydrolysis that may staining or calcium and phosphate from
occur during the 9-day incubation with incorporation at the 1 and 3 mM doses of the http
alkaline phosphatase is not sufficient to inhibitor. Figure 2 also shows that the ://w
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produce a detectable increase in total ionic endogenous alkaline phosphatase activity in w
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phosphate levels in serum. serum is linked to serum’s ability to calcify c
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An additional experiment was collagen: the same dose of 1,10- brg/
carried out in order to determine whether the phenanthroline that irreversibly inactivates y g
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addition of alkaline phosphatase accelerates TNAP activity in a serum aliquot also es
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the calcification of other collagenous prevents bone re-calcification in the same n J
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matrices. This experiment used a 36 h serum aliquot. nu
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incubation time, since the kinetic experiment Figure B (Supplemental Data) ry 2
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(Figure 1) showed that there is uptake of shows that the re-calcification of bone in , 2
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serum calcium by bone at this time only if serum is dose dependently inhibited by 9
alkaline phosphatase has been added to beryllium, with no evidence of Alizarin
serum. As seen in Supplemental Figure A, staining or calcium and phosphate
rat tail tendons and demineralized newborn incorporation at beryllium doses of 3 µM
rat tibias stained positively for calcification and above. Beryllium inhibition of TNAP is
with Alizarin red after incubation for 36 h in competitive, with a Ki = 0.45 µM (26), and
serum plus alkaline phosphatase, but not in standard TNAP assays 50% inhibition of
after incubation for 36 h in serum alone. TNAP activity is consistently observed at 1
Chemical analyses showed that there was a µM beryllium (29,30). The fact that
significant amount of calcium and phosphate comparable, 1 µM doses of beryllium cause
in each matrix following the 36 h incubation 50% inhibition of bone re-calcification in
in serum plus alkaline phosphatase, and no serum (Figure B) suggests that the ability of
detectable calcium or phosphate in the same beryllium to inhibit serum-induced
matrices after incubation for 36 h in serum calcification is due to its ability to inhibit
alone (not shown). These experiments TNAP 2. Although a number of mammalian
7
enzymes have been tested for sensitivity to the subsequent 5 day incubation in DMEM.
inhibition by beryllium (30), to our Alkaline phosphatase inhibitors therefore
knowledge there are no mammalian serum prevent the serum-dependent initiation of
enzymes other than alkaline phosphatase bone re-calcification, not the subsequent
that are inhibited by micromolar beryllium growth of the initial mineral nuclei.
concentrations. Evidence that serum calcification
Figure C (Supplemental Data) activity is inhibited by TNAP depletion
shows that bone re-calcification is dose- and restored by the addition of
dependently inhibited by levamisole, an physiological levels of TNAP. We next
uncompetitive alkaline phosphatase inhibitor devised a method to prepare TNAP-depleted
(27,28). There is 50% inhibition of bone re- serum in order to further explore the role of
calcification in serum containing 1mM TNAP in the serum-induced calcification of
levamisole, and complete inhibition of bone collagen. In this procedure, the endogenous
re-calcification at 10 and 30 mM levamisole. alkaline phosphatase activity of serum is
Previous studies have shown that first irreversibly inactivated by treatment
comparable concentrations of levamisole are with the zinc chelator 1,10-phenanthroline,
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required for inhibition of TNAP activity in and the zinc-phenanthroline complex is then ow
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the epiphyseal plate cartilage of rachitic rats removed by dialysis against DMEM (which lo
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(31). does not contain Zinc). The resulting ed
An additional experiment was TNAP-depleted serum proved to have less from
carried out in order to verify that alkaline than 1% of the initial alkaline phosphatase http
phosphatase inhibitors prevent the initiation activity (Table 3) and is no longer able to ://w
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of bone re-calcification, not the subsequent induce the calcification of purified type 1 w
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growth of the initial mineral nuclei. The collagen (Figure 4) or of demineralized c
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design of this experiment is based on a newborn rat tibia (Figure 5). brg/
previous study which showed that The alkaline phosphatase activity of y g
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incubation of demineralized bone in serum newborn calf serum is 0.2 U/ml. To confirm es
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for 24 h is sufficient to cause the treated the essential role of TNAP in the serum- n J
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bone to completely re-calcify during induced calcification of collagen, we nu
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subsequent incubation in DMEM, but not therefore added 0.2 U/ml of purified TNAP ry 2
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sufficient for Alizarin red staining or to TNAP-deficient serum (Table 3). This , 2
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detectable levels of calcium and phosphate addition restored the ability of serum to 9
incorporation (3). The effects of inhibitors induce the calcification of purified type 1
on the serum-dependent formation of crystal collagen (Figure 4) and demineralized
nuclei within the matrix could therefore be newborn rat tibias (Figure 5).
evaluated by adding the inhibitors only to A control experiment was carried out
serum during the first 24 h incubation, while to address the possibility that there might be
the effects of the inhibitors on the non-protein-associated zinc in the TNAP
subsequent growth of these nuclei could be solution, that this available zinc might
assessed by adding the inhibitors only to activate a zinc-dependent protein in serum
DMEM during the 5 d incubation. Figure 3 other than TNAP, and that the activation of
shows that each of the three alkaline this protein might actually be responsible for
phosphatase inhibitors tested inhibited the the ability of the TNAP solution to restore
initiation of bone calcification during the the calcification activity of phenanthroline-
first 24 h in serum, but did not inhibit the treated serum. The TNAP solution was first
growth of the initial mineral nuclei during dialyzed exhaustively against DMEM using
8
a 6-8 kDa MWCO membrane in order to grow, however. Because the vast majority
remove any non-protein-associated zinc; this of the nuclei are in the solution outside of
dialysis step did not significantly diminish the fiber, the only mineral formed in
TNAP activity. In a repeat of the amounts that can be detected is the mineral
experiment shown in Figure 4, we added 0.2 precipitate found on the bottom of the well,
U/ml of the dialyzed TNAP to TNAP- not mineral within the fibril (6).
deficient serum. The results of this In the present investigations, we
experiment were comparable to those shown have used fetuin-depleted serum to directly
in Figure 4: the addition of dialyzed TNAP evaluate the possible role of TNAP in the
fully restored the ability of serum to induce formation of apatite crystals in serum that
the calcification of purified type 1 collagen contains no collagen matrix. Fetuin was
(not shown). again removed from bovine serum using
Taken together, these observations rabbit anti- bovine fetuin antibodies; the
provide convincing evidence that TNAP fetuin content of the resulting fetuin-
plays an essential role in the serum-induced depleted serum was over 1000-fold lower
calcification of collagen. than the fetuin content of the original bovine
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The effect of alkaline phosphatase serum (Supplemental Table A). Figure 6 ow
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and its inhibitors on the formation of shows that the addition of TNAP more than lo
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apatite crystals in fetuin-deficient serum doubled the rate at which calcium is ed
that contains no collagen. In the course of incorporated into crystals during incubation from
the present study, we discovered that of DMEM containing 10% fetuin-depleted http
removing fetuin from serum eliminates the serum at 37°C, with half maximal calcium ://w
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ability of serum to induce the calcification incorporation at 15 h in fetuin-depleted w
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of a type I collagen matrix, and that adding serum plus TNAP compared to 38 h in c
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purified fetuin to fetuin-depleted serum fetuin-depleted serum alone. The addition of brg/
restores this activity (6). We further found TNAP also reduced the incubation time y g
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that a massive mineral precipitate forms needed for the first visible evidence of es
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during the incubation of fetuin-depleted crystal formation in this experiment n J
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serum, but not during the incubation of (cloudiness), from 30 hours in the absence nu
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serum containing fetuin (6). This mineral of TNAP to 12 hours in the presence. In ry 2
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precipitate forms whether or not a collagen agreement with previous studies(6), a , 2
0
1
matrix is present. These observations are massive precipitate could be seen in all 9
consistent with the hypothesis that a large wells by 48h and chemical analysis of this
serum nucleator generates apatite crystals in precipitate again confirmed that it is a
the solution outside of the collagen fibril, calcium phosphate mineral (not shown).
some of which penetrate into the aqueous It is of interest to note that calcium is
interior of the fibril (6). Since fetuin can incorporated into mineral at a significantly
only trap those nuclei that it can access, the faster rate in fetuin-depleted serum (t =
1/2
crystal nuclei that penetrate the fibril grow 38h, Figure 6) than in fetuin-replete serum
far more rapidly than those nuclei trapped (t = 60h, Figure 1). While the addition of
1/2
by fetuin outside of the fibril, and the TNAP accelerates calcium incorporation
collagen fibril therefore selectively calcifies. into mineral in both solutions, the rate of
When fetuin is removed from serum, the calcium incorporation into mineral in fetuin-
same number of mineral nuclei still form, depleted serum plus TNAP (t = 15h,
1/2
and some of these no doubt still penetrate Figure 6) remains substantially faster than
the fibril. All crystal nuclei are now free to in fetuin-replete serum plus TNAP (t =
1/2
9
38h, Figure 1). It seems probable that the DISCUSSION
relatively faster growth of mineral in fetuin- The present experiments were
depleted serum is in part due to the fact that carried out to determine whether tissue-
the crystals grow in the solution, while the nonspecific alkaline phosphatase is a
mineral in fetuin-replete serum must grow component of the serum activity that
within the collagen fibrils of the decalcified calcifies type I collagen. The results of
bone. The constraints imposed by the these tests show that TNAP plays a critical
location of crystals within the collagen role in the calcification of type I collagen in
fibrils are likely to substantially reduce the serum, and that this role is to activate a
overall growth of mineral in the case of serum nucleator of apatite crystal formation.
fetuin-replete serum. Because of the potential importance of these
It is also of interest that the addition results to an understanding of normal
of purified TNAP to serum does not produce biomineralization, it is important to begin
a proportional, 50-fold increase in the rate of with a discussion of the experimental
collagen calcification in fetuin-replete serum evidence.
(Figure 1) or in the rate of apatite crystal The first experiments showed that
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formation in fetuin-deficient serum (Figure the addition of purified alkaline phosphatase ow
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6). This observation indicates that there are to serum consistently accelerates the lo
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other steps in the serum-induced apatite calcification of demineralized bone, a ed
crystal formation pathway that become rate collagenous matrix that was once calcified, from
limiting at high TNAP doses. and rat tail tendon collagen, a collagenous http
An additional experiment was tissue that does not normally calcify in vivo. ://w
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carried out to further examine the effects of These observations show that the addition of w
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TNAP and its inhibitors on the formation of alkaline phosphatase does not accelerate the c
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apatite crystals in fetuin-depleted serum. As calcification of matrices by virtue of its brg/
seen in Figure 7, the formation of a mineral action on a matrix component that is unique y g
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phase in DMEM containing 10% fetuin- to tissues that normally calcify, and suggests es
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depleted serum was completely prevented that TNAP may instead accelerate matrix n J
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by 30 µM beryllium sulfate, 30 mM calcification by acting on a component of nu
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levamisole, or 3 mM 1,10-phenanthroline. serum that drives mineral formation. ry 2
2
This complete inhibition is remarkable, as TNAP is a phosphomonoesterase, , 2
0
1
the incubation was for 6 days while only 2 and a potentially trivial explanation for the 9
days are needed for mineral formation in the ability of alkaline phosphatase to accelerate
absence of inhibitor (Figure 6). Figure 7 collagen calcification in serum is that the
also shows that the addition of TNAP to enzyme could liberate sufficient phosphate
DMEM is not sufficient for the formation of from serum phosphomonoester substrates to
a mineral phase in the absence of fetuin- increase total ionic phosphate levels, and
depleted serum. We were also unable to that this increase in serum phosphate could
achieve the calcification of a type 1 collagen accelerate the re-calcification of
matrix using DMEM plus TNAP, DMEM demineralized bone. This possibility was
plus fetuin, or DMEM plus TNAP and fetuin tested by measuring ionic phosphate levels
(not shown). These observations in serum before TNAP addition, and after
demonstrate that crystal generation requires addition of TNAP and incubation for 9 days
a serum component other than TNAP or at 37°C, and no significant increase in
fetuin. phosphate was observed (Table 2). We
conclude that added TNAP does not increase
10
the rate of collagen calcification by raising additional studies to determine the effect of
serum phosphate levels. alkaline phosphatase and its inhibitors on the
The next experiments showed that formation of apatite crystals in fetuin-
three inhibitors of tissue non-specific deficient serum. These experiments showed
alkaline phosphatase each prevent the that adding purified bovine TNAP to fetuin-
calcification of collagen in serum. In each deficient serum approximately doubled the
case, the inhibitor dose required for rate of crystal formation (Figure 6). Doses
inhibition of serum-induced calcification is of TNAP inhibitors that prevent the re-
comparable to the dose previously found to calcification of demineralized bone also
inhibit purified TNAP in vitro. For the prevented the formation of crystals in fetuin-
irreversible inhibitor 1,10-phenanthroline, deficient serum (Figure 7). Since TNAP
there was also an excellent correlation did not cause crystals to form in the absence
between the inhibition of alkaline of fetuin-depleted serum, TNAP must act on
phosphatase activity in a serum aliquot and a serum component (the serum nucleator) to
the reduction in serum-induced collagen induce crystal formation.
calcification in the same aliquot (Figure 2). A molecular mechanism for the
D
In a final test, the alkaline calcification of collagen matrices. A ow
n
phosphatase activity of serum was critical problem in biomineralization is to lo
a
d
inactivated by treatment with the zinc understand how a matrix is mineralized. ed
chelator 1,10-phenanthroline, and the zinc- With the present study, we have now from
phenanthroline complex was then removed partially solved this problem for the http
by dialysis (Table 3). As anticipated, the calcification of the type 1 collagen fibril in ://w
w
resulting TNAP activity-deficient serum was neat serum (6,7). To our knowledge, these w
.jb
unable to calcify either purified type 1 are the first studies that provide a defined c
.o
collagen (Figure 4) or demineralized bone molecular explanation for matrix brg/
(Figure 5). However, if the normal mineralization. y g
u
physiological level of purified TNAP was The molecular mechanism that es
t o
added to this TNAP-deficient serum (Table selectively calcifies collagen fibrils in serum n J
a
3), the ability to calcify collagen was requires four critical elements (Figure 8): nu
a
completely restored. These observations 1. A matrix with an interior aqueous ry 2
2
demonstrate that phenanthroline prevents the compartment that is accessible to small , 2
0
1
calcification of collagen in serum by virtue apatite crystals but not large molecules. The 9
of its ability to inactivate TNAP, and show collagen fibril is 70% water by volume (8),
that the addition of physiological levels of and during calcification the water within the
purified TNAP to serum is sufficient to fibril is replaced by mineral (10,11). We
restore the ability of TNAP-deficient serum have recently discovered that all of the water
to calcify collagen. within the uncalcified fibril is in rapid
Evidence that tissue non-specific diffusion equilibrium with molecules up to
alkaline phosphatase is required for the the size of a 6kDa protein, and completely
formation of apatite crystals in fetuin- inaccessible to molecules larger than a
deficient serum. In the course of these 40kDa protein (7). Hydroxyapatite crystals
studies, we discovered that removing fetuin up to the size of a 6 kDa protein (about 12
from serum causes the formation of unit cells (7)) are therefore in principle able
numerous apatite-like crystals; these crystals to attain rapid diffusion equilibrium with the
form whether or not a collagen matrix is entire volume of water within the collagen
present (6). We therefore carried out