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J. Biol. Chem., Vol. 277, Issue 7, 5126-5133, February 15, 2002
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From the
Received for publication, August 16, 2001, and in revised form, November 15, 2001
Matrix vesicles are lipid bilayer-enclosed
structures that initiate extracellular mineral formation. Little
attention has been given to how newly formed mineral interacts with the
lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the
membrane is involved, we analyzed changes in lipid composition and
extractability during vesicle-induced calcification. Isolated matrix
vesicles were incubated in synthetic cartilage lymph to induce mineral
formation. At various times, samples of the lipids were taken for
analysis, extracted both before and after demineralization to remove
deposited mineral. Phosphatidylserine and phosphatidylinositol both
rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after
demineralization revealed that phosphatidylserine had become complexed
with newly forming mineral. Concomitantly, its levels actually
increased, apparently by base-exchange with phosphatidylethanolamine.
Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and
phosphatidylethanolamine also underwent rapid breakdown, but
phosphatidylcholine was degraded more slowly, all accompanied by a
buildup of free fatty acids. The data indicate that phosphatidylserine
forms complexes that accompany mineral formation, while degradation of
other membrane phospholipids apparently enables egress of crystalline
mineral from the vesicle lumen.
Matrix vesicles (MV)1
are extracellular microstructures released by calcifying cells that
initiate mineral formation in newly forming bone (1-4). MV are
enclosed by a lipid bilayer membrane that is enriched in selected
phospholipids, especially phosphatidylserine (PS) (5-6), a lipid with
known high affinity for Ca2+ (7-8). Initially, PS is
largely confined to the inner leaflet of the MV membrane (9). Previous
transmission electron microscopic (TEM) studies have shown that the
first mineral formed in MV is associated with the inner aspect of the
membrane (10). Later, the crystals appear to emerge through the
membrane and trigger formation of radial clusters of mineral centered
on the remnant of the original vesicle. How the crystals penetrate the
MV membrane is currently unknown. While it is possible that simple
physical force mediates this process (11), there is indirect evidence that latent lipolytic enzymes become activated during Ca2+
accumulation and facilitate breakdown of the membrane. To explore this
latter possibility, the composition of lipids in MV was analyzed during
the course of MV-mediated mineralization in vitro.
Since previous work had shown that not all lipids were readily
extracted from mineralizing tissues (12-13), resident MV lipids were
extracted both before and after demineralization using both neutral and
acidic lipid solvents. Lipid composition was analyzed qualitatively by
high performance thin layer chromatography (HPTLC) and quantitatively
by high performance liquid chromatography (HPLC) using an evaporative
light scattering (ELS) detector, which enabled accurate quantitation of
the various lipids.
Our findings reveal that extensive phospholipid degradation occurred
during MV calcification, and this was accompanied by a concomitant rise
in the amount of free fatty acids (FFA) apparently released by
phospholipases present in the vesicles. The breakdown of MV
phospholipids was accompanied by a substantial reduction in the
extractability of certain phospholipids, and the composition of MV
lipids changed significantly during the process of mineralization. In
particular, PS, which became progressively more tightly complexed with
nascent mineral, could only be fully extracted after demineralization. It was not only protected from degradation, but was actually
synthesized, apparently by a base-exchange mechanism.
Isolation of Matrix Vesicles--
Large batches of
collagenase-released matrix vesicles (CRMV) were isolated from the
metatarsal growth plate cartilage of 6 Mineralization Experiments--
To have sufficient material for
accurate lipid analyses, the large-scale preparations of CRMV (~20 mg
of protein) were allowed to mineralize by incubation in 600 ml of SCL
at 37 °C. At each time point (0, 2, 4, 6, and 24 h) for lipid
analysis a 110-ml sample was centrifuged at 100,000 × g for 60 min to sediment the CRMV. The resulting pellets
were transferred to individual glass tubes for lipid extraction after
resuspension in ~150 µl of SCL. Mineral formation in samples of the
incubation mixture was monitored by light scattering, essentially as
described previously (14).
Lipid Extraction and Purification--
Lipids were extracted
from the CRMV pellets, essentially as previously described (18). For
qualitative HPTLC analyses, the vesicle pellets were extracted with
chloroform, methanol (2:1) (v/v) (~20 ml/ml aqueous medium),
followed by sonication for 1 HPTLC and HPLC Analysis of Lipids--
For qualitative analysis,
the pure lipids from the preceding steps were analyzed by HPTLC on
Whatman LHP-K silica gel plates as described previously (20, 21).
Mixtures containing 10 µg each of various lipid standards were
applied to separate lanes on the same plate as the MV lipid samples.
Lipids were visualized by spraying with cupric-phosphoric acid charring
reagent (10% CuSO4 in 8% H3PO4)
and heating at 180 °C for 10 min in an oven (20). HTLC plates were
photographed and digitized for semi-quantitative estimation of lipid levels.
For accurate quantitative analysis, the lipids were analyzed using a
Shimadzu HPLC (SCL-10A Systems Controller, SIL-10A Auto Injector, Dual
LC-10AT Liquid Chromatographs to provide the gradients). The HPLC
system was equipped with an Alltech Varex MKIII ELS detector system.
Lipids were separated on a Lichrosorb SI-100 4.6 × 250 mm, 10 µm particle size, HPLC column supplied by Alltech Inc. Injection
volume was 30 Calcification of Isolated CRMV--
Mineralization induced by the
isolated CRMV was monitored by formation of mineral from the SCL during
the incubation period. As had been previously observed, there was a
progressive induction of mineral formation during incubation of the
CRMV with SCL (Fig. 1A). As is
typical of MV mineralization, there was a short lag period (~1 h)
during which minimal Ca2+ accumulation occurred;
thereafter, the rate of mineral formation increased rapidly and by
6 h mineral formation was ~80% of maximum. Fig. 1B
shows an electron micrograph of early MV mineralization made after
2 h of incubation in SCL.
Lipid Composition of CRMV--
Three different methods of analysis
were used to assess the lipid composition of CRMV, both before and
after incubation with SCL, which induced mineral formation. Initially,
for qualitative analysis, HPTLC was used to analyze the various polar
and nonpolar lipid classes present in extracts of CRMV made before and
after demineralization. From these studies it was evident that
significant changes in lipid composition were occurring during MV
calcification. Most notable was the dramatic disappearance of PS and
phosphatidyl inositol (PI) in extracts made before decalcification
after only ~2 h of incubation (Fig.
2A). Also there were
progressive increases in the levels of FFA and in the band that
included free cholesterol (CH) and 1,2-diacylglycerols (DG). On the
other hand, there was a strikingly different lipid composition in
extracts made after decalcification (Fig. 2B). Here there
was a dramatic increase in the levels of PS at time points when MV
calcification was in rapid progress.
While densitometric scans of the HPTLC plates afforded reasonable
estimation of the levels of individual lipids present, we used HPLC to
more accurately measure the quantitative changes in lipid composition.
Initially, we employed a highly sensitive UV detection system at 205 nm
to quantitate lipids present (22). But because of the variability in
sensitivity to individual lipids, which depended on the degree of fatty
acid unsaturation, we found this HPLC technique of limited utility.
With careful calibration, some useful information was obtained.
However, ELS detection proved to be the method of choice for
quantitation of the individual lipid classes, since it was not
dependent on chromophores. Shown in Table
I are the percentages of the total
phospholipid of various lipid classes in combined extracts made before
and after decalcification at successive time points from
T0 to T24.
In general agreement with previous analyses (23-24), at
T0, phosphatidylcholine (PC) was the major
phospholipid in MV, representing about 50% of the total.
Phosphatidylethanolamine (PE), ~17%, PS ~9%, sphingomyelin
(SPH), ~8%, monoacyl (i.e. lyso)phosphatidylethanolamine (LPE), ~6%, and PI, ~4% were the principal lipids. Low levels of
lysophosphatidylinositol (LPI), 1.4%, lysophosphatidylcholine (LPC),
1.2%, and lysophosphatidylserine (LPS), 0.05% were the only other
phospholipids consistently detected. With time, in each successive set
of extracts, the lipid composition progressively changed. The most
notable changes were the significant increases in the levels of PS,
LPS, and LPE and the progressive disappearance of both PI and SPH
(Table I).
However, these values were based on the analyses of lipids present in
extracts taken at the successive points during MV calcification. During
this period, changes in lipid composition were occurring due to both
variable lipid degradation and to specific effects on extractability
caused by the selective binding of certain lipids to the newly forming
mineral. Because of this, an attempt was made to normalize the
progressive changes occurring in total lipid composition by combining
data on total polar and nonpolar lipids from extracts taken before and
after decalcification at T0 to serve as a
baseline for changes in individual lipid classes occurring at
subsequent time points. These changes were then expressed as a
percentage of the original amount of each lipid class at successive time points.
Changes in Total Lipid Content--
Combining data on
lipids extractable before and after demineralization at each time point
caused only modest (±10%) overall change in the total lipid
(phospholipid + nonpolar lipid) content to occur during
MV-induced calcification. However, when the fate of the total
phospholipid was separated from that of the total nonpolar lipid, major
progressive changes in each type of lipid were evident (Fig.
3A). Whereas the amount of
total phospholipid underwent rapid and progressive decline, the
opposite occurred with the nonpolar lipid. After 6 h of
incubation, only about 50% of the original phospholipid remained,
whereas a nearly 33% increase over the original amount of nonpolar
lipid was seen. By 24 h, only about 20% of the total phospholipid
remained, whereas the amount of nonpolar lipid had increased 66% above
its original level.
Examination of the combined data on individual lipids extractable
before or after demineralization revealed major differences in the
stability of each class of phospholipid (Table
II). These revealed that rapid
degradation of the acidic phospholipids PS and PI occurred; for both
lipids only about 50% of the original remained after 2 h of
incubation. However, surprisingly, PS "recovered" and returned to
nearly 75% of the original by 24 h. In contrast, levels of PI
continued their rapid decline and by 24 h, less than 10% of the
original lipid remained. SPH also showed very rapid and continuous
degradation, whereas breakdown of PE and PC was significantly slower.
The changes in the content of lysophospholipids were complex and will
be elaborated on later. However, overall the HPLC findings corroborate
and clarify the qualitative impression observed upon HPTLC
analyses.
Changes in Extractability of Total Lipids--
As MV calcification
progressed with time, the percentage of the total phospholipid
extractable before demineralization decreased concomitantly, and
correspondingly, the proportion extractable only after demineralization
increased (Fig. 3B). At T0, nearly 95% of the total phospholipid present was extractable before
demineralization; however, after 4 h of incubation, by which time
mineralization was well in progress, less than 80% was extractable. By
24 h, only about 70% of the remaining phospholipid could be
extracted prior to demineralization. This indicated that with
calcification, progressively more of the surviving phospholipid was
bound to the mineral and could not be extracted until the vesicles were demineralized. In contrast, the extractability of the nonpolar lipid
was much less affected by mineralization. At T0,
about 90% of the nonpolar lipid was extractable before
demineralization. With time this increased to about 93-94% (data not shown).
Changes in Extractability and Survival of Individual MV
Lipids--
Corroborating the impression gained by HPTLC, analysis by
HPLC revealed that MV mineralization had a profound effect on the extractability and survival of the two principal acidic phospholipids, PS and PI. At T0 before exposure to the
mineralizing solution, about 90% of the PS was extractable before
demineralization; however, after only 2 h of incubation, only 5%
of the original PS was extractable (Fig.
4A). There was a corresponding
major increase in the proportion of the original PS that survived and
could be extracted only after demineralization. This increased from
~10% at T0 to about 75% at
T4 and remained at this level thereafter.
A similar pattern of extractability was seen with PI, but as noted in
Table II, there was major degradation of this lipid during MV
mineralization. At T0, again about 90% of the
PI was extractable, but after 2 h of incubation, in contrast to
PS, almost one-third of the original PI could still be extracted (Fig.
4B). However, by T4 and thereafter,
no PI was extractable before demineralization. Again, in contrast to
PS, there was much less of the original PI that could be extracted
after demineralization. This value rose from ~7% at
T0 to a maximum of only about 18-20% of the
original at T4, decreasing gradually thereafter.
This indicated that much less of the original PI became complexed and
stabilized by the newly forming mineral and thus was susceptible to degradation.
In contrast to the acidic phospholipids, mineralization had only
a minor effect on the extractability of the neutral phospholipids. For
PE, about 90% of the original was extractable before demineralization at T0. The remaining 10% was apparently
complexed with the mineral and not extractable (Fig. 4C) and
was protected from degradation, persisting throughout the 24 h of
incubation. However, the amount of PE that was not complexed rapidly
decreased, indicating that it was rapidly degraded.
Essentially all (>95%) of the choline-containing phospholipids, PC
and SPH, were extractable before demineralization, regardless of the
length of incubation or extent of mineralization. Fig. 4D
reveals that PC was readily extractable before demineralization; however, its rate of degradation was slower than most other
phospholipids, remaining essentially linear throughout the incubation.
By 24 h, only about 20% of the original PC remained. Fig.
4E shows that SPH, also unprotected by complexation with the
mineral, was rapidly degraded. By 4 h, only about one-third of the
original SPH remained, and by 24 h there was only about 5% of the
original left. Thus, in contrast to all of the other diacyl
phospholipids, PS largely survived being protected by the newly forming
mineral and resynthesized from other lipids (see later).
Changes in Lysophospholipids--
Evident in all lipid extracts of
MV were significant amounts of lysophospholipids. LPE, the monoacyl
derivative of PE, was the most abundant form representing ~6% of the
total phospholipid for most of the incubation (Table I). As evident
from Fig. 4F, almost 95% of the LPE present initially was
extractable before demineralization. However this rapidly declined; by
4 h only about one-third, and by 24 h less than 20% of the
original LPE could be so extracted. On the other hand, the amount of
LPE not extractable until after demineralization increased
progressively during MV calcification, rising from ~5% at
T0 to ~35% at T24. The
other lyso-derivatives detected during MV calcification were LPI, LPC, and LPS. Small amounts of LPI and LPC (1.4 and 1.2% of the total PL,
respectively) were present initially, but there were only trace amounts
of LPS (Table I). While levels of LPI and LPC generally decreased
thereafter, levels of LPS increased. At 4 h, in lipids extractable
after decalcification, levels of LPS were dramatically increased (Fig.
4G). This corresponds to the time when the diacyl form, PS,
was no longer detectable in extracts made before demineralization (Fig.
4A). Expressed as a percentage of the original LPS present, it is evident that net synthesis of this lipid had occurred, probably in part from phospholipase A action on PS, but also from base-exchange of serine with LPE (see "Discussion").
Changes in Nonpolar Lipids--
Further insight into the
lipid degradation process came from analyses of the nonpolar lipids.
Initially at T0, CH was the dominant nonpolar
lipid accounting for ~40% of the total; its esterified form, CH
esters represented some 28-30%, with free fatty acids FFA and
triacylglycerols each accounting for ~9% of the total nonpolar
lipid. DG accounted for only 1.3% and MG accounted for <0.5% of the
total. Three uncharacterized, more polar nonpolar lipids accounted for
the remaining 10%. Based on HPLC analyses, the levels of total
nonpolar lipids in the successive extracts of MV during mineralization
rose significantly (Fig. 3A). HPTLC analyses (Fig.
2A) indicated that the levels of FFA and to a lesser extent,
1,2-DG (+CH) increased, whereas levels of 1,3-DG decreased with time.
Analyses of these data confirmed that FFA levels do indeed rise during
MV incubation (Fig. 5). While levels of
1,2-DG (+CH) were relatively unchanged, levels of 1,3-DG declined.
Lipids are integral components of the MV membrane that provides
the barrier to confine the contents of the vesicles. MV contain significant levels of internal Ca2+ and Pi
(15), which comprise key components of the nucleational core previously
demonstrated to be critical for mineral formation (14, 21). Studies by
Eanes et al. with synthetic models of MV (11) have revealed
that PS-rich liposomes with lipid composition similar to that of MV
block the emergence of mineral (25). On the other hand, TEM of
mineralizing MV has shown that the first crystals form in association
with the vesicle membrane and thereafter appear to penetrate through
the membrane (10). Since the primary function of MV appears to be the
induction of mineral formation, which membrane lipids bind to the
mineral? Also, what mechanism enables egress of the mineral to
propagate extravesicular mineralization?
While there is substantial evidence for the binding of mineral to
acidic phospholipids in MV (13, 23), for the presence of phospholipase
activity in growth plate cartilage (26), and in MV in particular (5,
27), there have been no studies of the changes in lipid extractability
and composition that occur when MV are allowed to mineralize under
controlled conditions in vitro. Such studies should provide
insight into how these aspects of MV mineralization are regulated.
Kinetic studies of MV-induced mineralization typically reveal a lag
period during which minimal Ca2+ accumulation occurs (28,
29), followed by a rapid uptake period, and finally a slower
plateau phase during which apatitic crystalline mineral forms (29, 30).
Studies on nascent MV reveal the presence of a nucleational core that
contains PS complexed with non-crystalline calcium phosphate (14).
Fourier-transform infrared studies indicate that the rapid uptake
period coincides with the formation of an octacalcium phosphate-like
crystalline phase (31). It is speculated that breakdown of the MV
membrane is what triggers this critical event.
To address these issues, we performed detailed analysis of the changes
in lipid composition that accompany the onset and progression of MV
mineral deposition. Our initial studies using HPTLC (Fig. 2) revealed
two major features: first, a marked change in the extractability of the
certain phospholipids, and second, the progressive breakdown of most MV
phospholipids accompanied by the accumulation of FFA. These studies
were confirmed and extended with HPLC analyses.
The decline in phospholipids appears to be due to the action of
various phospholipases that degraded these lipids releasing FFA and
1,2-DG), thereby increasing the overall nonpolar lipid level.
Which enzymes caused the degradation of the phospholipids that occurred
during the mineralization process? Insight has come from consideration
of the partial degradation products of these lipids. Action of
phospholipase A(1 or 2) on diacyl phospholipids would be
the corresponding monoacyl (i.e. lyso) forms; action of
phospholipase C would yield 1,2-DG and the corresponding phosphoryl base (e.g. phosphoryl choline). Subsequent action of other
lipases on lysophospholipids and DG would yield FFA and their
corresponding water-soluble products.
Since LPE could be generated from the action of phospholipase A(1
or 2) on PE, and the levels of PE rapidly declined during MV
mineralization, it is evident that both formation and degradation of
LPE must have been occurring concomitantly at a significant rate. The
presence of substantial amounts of LPE in the lipid extracts indicates
that there must be substantial phospholipase A(1 or 2)
activity in MV. Comparison of Fig. 4, B and F
indicates that the rate of LPE degradation was even greater than that
of PE. One might assume that lysophospholipase A activity in MV was even greater than phospholipase A activity, which is in agreement with
the lack of accumulation of LPC, despite the fact that PC levels
steadily declined. A more likely possibility is that LPE was being
converted to LPS by a base-exchange pathway with serine (see later).
Alternatively, PC may have been broken down by action of PLC (32),
which would yield 1,2-DG. Indeed, there appeared to be some
accumulation of 1,2-DG (Fig. 2A), but evidently it also was
degraded by a lipase to MG, which appeared to increase during MV
incubation. PC also may have been degraded by phospholipase D (33), but
there was no indication of an accumulation of phosphatidate.
Another interesting feature of lipid breakdown during MV
calcification was the rapid and almost complete loss of SPH. Classical sphingomyelinases cleave SPH, releasing ceramide and phosphoryl choline
(34). Recent studies indicate that ceramide enhances apoptosis and
breakdown of rabbit articular cartilage (35). Thus, this pathway of MV
lipid breakdown could contribute to vascular invasion of the growth
plate. Unfortunately, we did not analyze for the presence of ceramide
in our samples.
Comment needs to be made concerning the marked changes in
extractability of certain phospholipids, particularly PS, during MV
mineralization. PS is known to complex with Ca2+ (7, 8),
particularly in the presence of Pi, which favors the
formation of PS·Ca·Pi complexes (36). Indeed, after
2 h of incubation, PS could be extracted only after
demineralization. What was the state of PS in MV before exposure to SCL
and the onset of calcification? At T0 before
demineralization PS was almost fully extractable. This would seem to
indicate that it was not complexed with Ca2+ and
Pi. However, looking back to the original discovery of the PS·Ca·Pi complexes, it is evident that PS
complexed with amorphous calcium phosphate is quite soluble in
chloroform,methanol (36). Indeed, the data indicate that only after the
Ca2+·Pi mineral becomes crystalline
does the associated PS become non-extractable prior to demineralization.
Another phenomenon that at first seemed improbable was the discovery
that total PS levels in the MV actually rose between T2 and T4-24 (Table II).
How can this be, knowing that MV lack the ability to produce ATP needed
to drive classical phospholipid synthesis? The answer comes from the
demonstrated presence of the base-exchange pathway in MV (24). This
energy-independent pathway depends on an enzyme that exchanges
ethanolamine for serine in both monoacyl and diacyl forms of PE. Our
past studies with growth plate chondrocytes have shown that LPE is the
primary recipient of serine and forms LPS. This would explain the
striking rise in levels of LPS at T4 during MV
mineralization (Table II, Fig. 4G). It is apparent that PE
itself was also subject to this base-exchange pathway. This would
contribute to the major loss of this lipid and the net synthesis of PS
during MV mineralization. PS, thus, is not only protected from
degradation by the newly forming mineral, but its level becomes
augmented by conversion of PE to PS. Since the PS·Ca·Pi
complex is known to stimulate mineral formation (37), the conversion of
PE to PS itself may facilitate the mineralization process.
What type of phospholipase A activity is predominant in MV,
A1, or A2? A clue comes from studies in which
we attempted to quantitate the levels of MV lipids by HPLC using UV
absorption at 205 nm. Absorptivity at that wavelength is dependent on
the presence of double bonds characteristic of fatty acids present at
position 2 of glycerol in phospholipids. In our analyses of MV
phospholipids, LPE showed strong absorptivity at 205 nm indicative of
the presence of unsaturated fatty acids. If these reside primarily on
C-2 of the glycerol moiety of this phospholipid, then the LPE must have
arisen from action of phospholipase A1 and not
phospholipase A2. Obviously, this needs to be explored by
direct analyses of the constituent fatty acids of LPE, but this is
beyond the scope of this study.
We thank T. K Yoshimori for assistance in the
initial stages of the HPLC analyses and Stephen Welch for assistance in
the HPTLC analyses.
*
This work was supported by Grant AR18983 from the NIAMS,
National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, Univ. of South Carolina, 329 Graduate Science Research Center, Columbia, SC 29208. Tel.: 803-777-6626; Fax: 803-777-9521; E-mail: wuthier@mail.chem.sc.edu.
Published, JBC Papers in Press, November 19, 2001, DOI 10.1074/jbc.M107899200
The abbreviations used are:
MV, matrix vesicles;
PS, phosphatidylserine;
TEM, transmission electron microscopy;
HPTLC, high performance thin-layer chromatography;
HPLC, high performance
liquid chromatography;
ELS, evaporative light scattering;
FFA, free
fatty acids;
MG, monoacylglycerols;
CRMV, collagenase-released MV;
SCL, synthetic cartilage lymph;
PI, phosphatidylinositol;
CH, free
cholesterol;
DG, diacylglycerols;
PE, phosphatidylethanolamine;
PC, phosphatidylcholine;
SPH, sphingomyelin;
LPS, lysophosphatidylserine;
LPI, lysophosphatidylinositol;
LPE, lysophosphatidylethanolamine;
LPC, lysophosphatidylcholine.
Changes in Phospholipid Extractability and Composition Accompany
Mineralization of Chicken Growth Plate Cartilage Matrix Vesicles*
,,,¶
Department of Chemistry and Biochemistry,
University of South Carolina, Columbia, South Carolina 29208 and
the § Department of Pathology, McMaster University,
Hamilton, Ontario L8N 3Z5, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to 8-week-old broiler strain
chickens using previously described methods (14). In brief, cartilage
shavings from ~300 chicken feet (~160 g) were digested with 0.1%
trypsin (type III, Sigma) at 37 °C for 30 min in a synthetic
cartilage lymph (SCL) (14) with an ionic composition similar to that
found to be present in native cartilage (15). The trypsin solution was
removed; tissue slices were rinsed twice with SCL and digested with
collagenase (200 units/g of tissue, type IA, Sigma) at 37 °C for
33.5 h. The partially digested tissue was vortexed, and the
suspension centrifuged as previously reported to sediment the CRMV
(14). The pellet was resuspended as a stock containing 5.0 mg of
vesicle protein/ml in SCL modified to have one-half the normal level of Ca2+ to prevent dissolution of labile Ca2+ and
Pi (16) and also to minimize Ca2+ uptake by the
CRMV during storage. Protein levels were determined by the Lowry
et al. method (17).
2 min (extract 1). The tubes were then
centrifuged at 3,000 rpm for 12 min to sediment the insoluble residue.
Initially, after collecting the lipid-containing supernatant, a second
extraction with chloroform,methanol,HCl (200:100:1) (v/v/v) was
performed, assuming that any mineral-complexed would be readily
extractable. However, subsequent work revealed that significant amounts
of the acidic phospholipids remained in the residue. Therefore, for all
studies reported here, after the initial lipid extraction, the CRMV
pellets were then demineralized with 0.5 M sodium salt EDTA for 20 min at room temperature and sedimented by
centrifugation at 3,000 rpm for 12 min. After removal of the
supernatant, the decalcified residue was reextracted using
chloroform,methanol,HCl (200:100:1) (v/v/v) (extract 2), which was
found to quantitatively remove the remaining lipids. The crude extracts
were dried under N2 and partitioned through a Sephadex G-25
column to remove non-lipid contaminants (19).
50 µl (autosampler). The two mobile phases used were
as follows: A, methanol,water (80:20) (v/v); B,
chloroform,methanol,0.1% formic acid (80:20:0.1) (v/v/v). The
following gradient program was used: 1) 92% B,8% A for 6 min; 2) 92%
B,8% A to 56% B,44% A in 21 min; 3) 56% B,44% A to 20% B,80% A
in 2 min, 4) 20% B,80% A to 100% B in 1 min, 5) 100% B to 92%
B,8% A in 3 min. Total run time was 33 min, with a solvent flow rate
of 1 ml/min. Nitrogen gas, 11.6 pounds per square inch, was used as the
carrier at a flow rate of 2.02 standard liters per min in the ELS
detector, which was operated at 72 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
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Fig. 1.
Mineral phase induction by CRMV. MV
samples were incubated in SCL, and mineral formation was assessed by
light scattering at 340 nm (14) (A) and by TEM
(B). A, after a lag period of about 1 h,
rapid increase in the rate of mineralization ensued; after ~4 h the
rate of mineralization slowed significantly. B, TEM of
mineral formation assessed after 2 h incubation. At the
upper right is a MV containing little mineral, but with
membrane still intact. Immediately below and slightly to the
left is a MV, sectioned near its edge, with mineral
crystallites at its center and developing radially away in conjunction
with associated collagen type II fibrils. At lower left
center is an intensely mineralized MV with more advanced mineral
formation largely obscuring the original MV.
View larger version (72K):
[in a new window]
Fig. 2.
HPTLC analysis of lipids present in CRMV
after various times of incubation with SCL. A (extract
1), lipid composition of the CRMV extracts made with
chloroform,methanol (2:1) (v/v) before demineralization. B
(extract 2), lipid composition of CRMV extracts subsequently made with
chloroform,methanol,concentrated hydrochloric acid (200:100:1) (v/v/v)
after demineralization with EDTA. A, lane 1, standards in descending order: cholesterol esters (CHE),
free fatty acids (FFA), free cholesterol (CH),
glucosyl ceramide (GC), phosphatidylinositol
(PI), and sphingomyelin (SPH); lane 2, triacylglycerols (TG), 1,2-diacylglycerols
(1,2-DG), monoacylglycerols (MG),
phosphatidylethanolamine (PE), phosphatidylserine
(PS), phosphatidylcholine (PC); lane
3, lysophosphatidylethanolamine (LPE),
lysophosphatidylinositol (LPI), lysophosphatidylserine
(LPS), lysophosphatidylcholine (LPC); lanes
4 8, extract 1, lipids from CRMV incubated 0, 1.5, 3, 6, and
22 h, respectively. B, lane 1, standards in
descending order: CHE, FFA, CH, GC, PI, PS, and SPH; lane 2,
TG, 1,2-DG, MG, PE, and PC; lane 3, LPE, LPI, LPS, and LPC;
lanes 48, extract 2, lipids from CRMV incubated 0, 1.5, 3, 6, and 22 h, respectively. 1,3-Diacylglycerols (1,3-DG) appeared
as a minor band immediately below 1,2-DG in lane 2 of lipid
standards in both A and B.
Changes in matrix vesicle lipid composition during in vitro
calcification
View larger version (14K):
[in a new window]
Fig. 3.
Changes in content and extractability of
total phospholipid and nonpolar lipid fractions of CRMV during in
vitro calcification. Collagenase-released MV were
incubated in SCL as described under "Experimental Procedures," and
the total lipids extracted before (extract 1) and then after
demineralization with EDTA (extract 2) at each stage of incubation.
A, changes in the total lipid content, phospholipids
(closed symbols) and nonpolar lipids (open
symbols) from combined extracts 1 and 2, expressed as a percentage
of the total original lipid present in the vesicles. B,
changes in the extractability of total phospholipids extractable before
(extract 1, closed symbols) and after (extract 2, open
symbols), expressed as a percentage of the total phospholipid
present. Values are the mean ± S.E. of six separate
analyses.
Recovery of matrix vesicle lipids during in vitro calcification
View larger version (33K):
[in a new window]
Fig. 4.
Changes in the percentage of individual
phospholipid classes recoverable in extracts made before (closed
symbols) and after (open symbols)
demineralization. A, phosphatidylserine
(PS), B, phosphatidylinositol (PI),
C, phosphatidylethanolamine (PE), D,
phosphatidylcholine (PC), E, sphingomyelin
(SPH), F, lysophosphatidylethanolamine
(LPE), and G, lysophosphatidylserine
(LPS). Data presented are the percentages at each time point
during progressive stages of MV calcification of the original amount of
each phospholipid class recovered in extracts made before and after
demineralization, expressed as the mean ± S.E. The means are from
six separate analyses for all lipid classes except SPH, for which only
five analyses were available at 24 h and LPS, for which only three
analyses were available at all time points.
View larger version (14K):
[in a new window]
Fig. 5.
Changes in the percentage of three key
nonpolar lipid classes recoverable in extracts of CRMV made during
in vitro calcification. FFA,
closed circles; 1,2-DG + CH, open squares;
1,3-DG, open triangles. Data presented are the percentages
at each time point of the original amount of each nonpolar lipid class
recovered in extracts made before demineralization, expressed as the
mean ± S.E. of three estimates from HPTLC analyses.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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