An analysis of Ca2+ release
by DGEA: mobilization of two functionally distinct internal stores
in Saos-2 cells
T. J.
McCann1,2,
G.
Terranova2,
J. W.
Keyte3,
S. S.
Papaioannou1,
W. T.
Mason2,
M. C.
Meikle1, and
F.
McDonald1
1 Department of Orthodontics
and Paediatric Dentistry, United Medical and Dental Schools, London
SE1 9RT; 2 Department of
Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT;
and 3 Biopolymer Synthesis and
Analysis Unit, School of Biomedical Science, Queens Medical Centre,
Nottingham NG7 2UH, United Kingdom
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ABSTRACT |
Osteoblasts can be activated by their collagen matrix and in
particular the DGEA peptide motif. We have reported that DGEA is able
to activate Ca2+ signaling
pathways in the human osteoblast-like cell line, Saos-2, by a tyrosine
kinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, and
F. McDonald. Matrix Biol. 16:
271-280, 1997). In the present study, we show that this activity
is due to coupling of the signal to intracellular
Ca2+ stores, since the DGEA action
is not blocked by La3+ but is lost
when Ca2+ stores are depleted with
2 µM and blocked by 10 µM ryanodine. The activated stores also
differ functionally from those activated by thrombin, as blockade with
U-73122 obstructs only thrombin-activated Ca2+ release. We have shown that
the DGEA activity was not due to its high-charge density, since the two
acidic residues can be substituted with their uncharged homologues
(asparagine and glutamine) without significant loss of activity. This
was in turn measured by an adhesion assay that also demonstrated this
level of specificity. Furthermore, by constructing DGEA bound to FITC,
we have shown that DGEA binding was dependent on divalent cations. We
have also demonstrated that an intact actin cytoskeleton is not
required for Ca2+ activation by
inhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant role
in regulating osteoblast function via this peptide motif.
calcium signaling; type I collagen
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INTRODUCTION |
THE MECHANISM OF OSTEOBLAST adhesion to the
extracellular matrix (ECM) of bone is complex, and both Arg-Gly-Asp
(RGD)-dependent and RGD-independent integrin binding are utilized.
Evidence exists to support the hypothesis that adhesion to a collagen
substrate regulates osteoblast differentiation and function. For
example, peptides containing the RGD motif blocked mineralization by
rat osteoblasts, disrupted their morphology, and inhibited subsequent osteoclast development but had no influence on collagen synthesis in
these cells (6). It has also been shown that the organic matrix
actively regulates the phenotype of cultured rat osteoblasts. Growth of
UMR-201 preosteoblast cells on type I collagen altered the pattern of
gene expression induced by retinoic acid (18), whereas primary cultures
of fetal rat calvarial osteoblasts showed accelerated and enhanced
differentiation on the same substrate and mineralized their matrix
(11).
The major mechanism for this adhesion and subsequent cellular control
is via integrin-ECM protein interactions; human osteoblasts express a
variety of integrin subunits, including
1-3,5 and
1. Indeed, Dedhar (2) provided
data suggesting that integrins of the
1-family may play a role in the
induction of alkaline phosphatase expression by interleukin-1 in human
MG-63 osteosarcoma cells. The
1
1-
and
2
1-integrins
have been identified as the major adhesion receptors for collagen (9),
and it seems likely that the regulatory activity of collagen on
osteoblasts is mediated by one or both of these receptors.
The peptide motif that is recognized by
1
1
has not yet been described, although there may be possible
cross-recognition with motifs previously identified, such as the RGD
sequence for fibronectin. The
2
1-integrin
is reported to recognize a tetrapeptide motif [Asp-Gly-Glu-Ala
(DGEA)] from type I collagen (16). A cyanogen bromide-digested
collagen fragment (CB3) and peptides based on this motif inhibited
platelet adhesion to collagen but not to laminin or fibronectin (17).
In addition, the
2
1-integrin is expressed by cultured human osteoblasts (1, 8), and we have
previously reported that the DGEA peptide causes a
Ca2+ increase in human
osteoblast-like cells (Saos-2) (13).
The DGEA peptide sequence is very acidic and would carry a net charge
of
2 at neutral pH. It was a possibility that the
Ca2+ signaling activity we have
described was related to nonspecific charge effects, due to the acidic
nature of the peptide. This study examines whether peptide fragments,
structurally related to the DGEA sequence but with varying charge,
could activate the Ca2+ signaling
pathway in Saos-2 cells and attempts to determine whether the response
is specific to the DGEA motif. This study is also correlated with a
functional study to determine if there is an effect of the
modifications of DGEA on cell adhesion. Furthermore, we attempt to
assess the origin of the Ca2+
response, determine if this is functionally distinct from a seven transmembrane spanning receptor, and establish if interaction of the
DGEA motif requires an intact actin cytoskeleton.
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MATERIALS AND METHODS |
Fura 2-AM, cytochalasin B, U-73122, and ryanodine were from Calbiochem
(Nottingham, UK); LaCl3, collagen,
human
-thrombin, FITC, triisopropyl silane, and crystal violet were
from Sigma/Aldrich (Poole, UK). Peptides were prepared by manual solid
phase synthesis using standard F-moc chemistry (materials from
Novabiochem, Nottingham, UK). Purity was checked by reverse-phase HPLC
using a C18 column and a water/acetylnitrile gradient and by amino acid
analysis. 45CaCl2
was purchased from Amersham International (Bucks, UK). All reagents for
the manufacture of DGEA bound to a fluorescent indicator were from
Applied Biosystems (division of Perkin Elmer, Foster City, CA).
Cell Culture
Transformed human osteoblasts, derived from an osteosarcoma (Saos-2
cells; American Type Culture Collection), were chosen to ensure
uniformity of intracellular signaling pathways. Of the human cell lines
available, Saos-2 cells appear to be the most highly differentiated
osteoblast-like cells (15) and are capable of effecting matrix
mineralization in vitro (14). These cells do not show voltage-activated
Ca2+ channel activity (12). Cells
were grown in DMEM-nutrient mix F-12 (1:1) with 10% FCS and 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B
at 37°C in a humidified atmosphere of 5%
CO2 and passaged once per week.
For imaging, cells were plated on glass coverslips (22 mm diameter, 1.5 gauge; Fison's, Loughborough, UK) and used 2-7 days later. Cells
were incubated in serum-free medium for 18-24 h before use.
Ca2+ Imaging
Cells were washed with HEPES-buffered salt solution (composition in mM:
127 NaCl, 5 KCl, 2 MgCl2, 1.8 CaCl2, 0.5 NaHPO4, 5 NaHCO3, 10 D-glucose, and 10 HEPES, pH 7.3)
and loaded with 4 µM fura 2-AM in the same buffer, containing 0.04%
Pluronic F-127, for 45-60 min at room temperature. Cells were then
rinsed with buffer for at least 20 min before use. For fluorescence
imaging, cells were mounted in a heated chamber (37°C) on the stage
of a Nikon inverted epifluorescence microscope and viewed with a ×40, 1.3 numerical aperture quartz objective. The cells were
illuminated with light at 340 nm and 380 nm, alternately, and emitted
light of 510 nm was directed to the image plane of a small-area, cooled charge-coupled device camera. The excitation filter wheel and camera
read-out were controlled by a 486 personal computer using MiraCal
software (Life Science Resources, Cambridge, UK). Sequential images
were saved to the computer hard disk, and the images were ratioed and
analyzed using MiraCal. Background images for each wavelength were
subtracted from each cellular image before ratioing. Fura 2 fluorescence was calibrated to
Ca2+ concentration with 10 µM
ionomycin in the presence of 5 mM
CaCl2 for maximal fluorescence
ratio or with no added Ca2+ and 10 mM EGTA for minimal fluorescence ratio (7). Agonists were
added as twofold concentrated stock solutions to an area of the
coverslip remote from that being imaged.
Where comparisons were made of the number of cells responding, a
response was defined as at least a twofold increase in intracellular Ca2+ following addition of ligand,
compared with the mean basal level in that experiment.
45Ca Influx Into Cells
To analyze the possible blockade of
Ca2+ by
La3+ into cells, a series of
experiments was performed using 3 µCi of
45CaCl2.
Cells (105) were seeded into
four groups of four-well Labtek slides (GIBCO, Paisley, UK); two groups
had 3 µCi of
45CaCl2
added to the medium. After 3 days, the medium was discarded; the cells
were washed three times with PBS and then serum starved for 18 h. Four
groups of cells were arranged as indicated in Table 1 with
45Ca2+
in the medium of two groups that were subsequently stimulated with
DGEA-thrombin with or without
La3+. Thrombin activates a
well-characterized seven transmembrane receptor causing
Ca2+ to enter the cell (10). The
group of cells that had been incubated in the presence of
45Ca2+
acted as the baseline, although serum starvation did reduce the levels
of radioactivity detected within these cells. The cells were stopped at
30-s intervals after stimulation with DGEA or thrombin and washed four
times with 1-ml volumes of ice-cold balanced salt solution (in mM: 135 NaCl, 5.6 KCl, 1.2 MgSO4, 2.2 CaCl2, 10 glucose, and 20 HEPES-NaOH, pH 7.4). The intracellular
45Ca was extracted with 1% Triton
X-100 and measured using a scintillation counter in 10 ml of
scintillation fluid (23). The values were then expressed as a
percentage of the unstimulated cells at the beginning of the
observation period.
Cell Adhesion Assay
The basis of this assay was to read the optical density of an unknown
number of Saos-2 cells stained with crystal violet at 590 nm and
compare this with the known optical density of a specific cell number
(5). The experiments were established on 24-well plates grown on 1 mg/ml collagen and sterilized overnight under ultraviolet light. Cells
(105) were added to each well
together with 750 µM of the relevant peptide. The plates were stopped
at 1, 2, 4, 8, and 16 h by the addition of 1 ml of 1% formaldehyde in
PBS for 1 h. The dish was washed three times with PBS and then stained
for 1 h with 1 ml of 0.1% crystal violet. The cells were washed six
times with distilled water, air dried, and solubilized with 0.2%
Triton X-100. The plate was then read at 590 nm on a multiplate
reader.
Peptide Synthesis
The peptide was synthesized on an Applied Biosystems 431A instrument,
modified with a flow-through deprotection monitor. Standard FastMoc
chemistry cycles were used (user manual version 2, September 1991).
FITC was coupled to the resin-bound peptide, after the final piperidine
treatment, using 1.5 equivalents of FITC and 1.5 equivalents of
N,N-diisopropylethylamine.
Cleavage and deprotection of the final product were accomplished in
trifluoroacetic acid, water, and triisopropyl silane (94:5:1) for 2 h
at room temperature.
Saos-2 cells were plated on four-well Labtek slides (50,000 cells/chamber) and grown to confluence (2-3 days). Each chamber then had the appropriate external medium applied. The conditions of
DGEA binding examined were 1)
Ca2+ removed by the addition of
EGTA (2 mM), 2)
Ca2+ replaced by
Ba2+ (30 nM),
3)
Ca2+ replaced by
La3+ (30 nM), and
4)
Ca2+ replaced by
Mg2+ (30 nM). The control was DGEA
bound to the cells but without FITC. After 4 min DGEA bound to FITC was
added to the cells, and after 90 s the cell were fixed in 4%
paraformaldehyde (pH 7.5) for 5 min. The cells were washed three times
with PBS for 5 min and mounted in Citofluor. Chambers were viewed under
a Nikon fluorescent microscope. For photomicrography, cells were
photographed using a ×40, 1.3 numerical aperture objective and a
Nikon F801 camera. Fields with fluorescence were photographed with
automatic exposure, and the control fields were exposed for the same
times.
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RESULTS AND DISCUSSION |
Structural Analysis of DGEA Peptide Sequence Binding to Cells
Variations of the DGEA sequence were used as agonists to the cells to
establish the roles of each amino acid in the activation of the
intracellular signaling pathway. It was clear that substitution of one
amino acid made a significant difference to the activation of the
peptide. The data in Fig. 1
(n = 20) show an
example of the Ca2+ response
elicited by DGEA, which is compared with a variant of this, DGAA, in
Saos-2 cells (n = 20, ±SE). DGEA
at 400 µM induced a response in all the cells in this particular
field. Figure 1 clearly shows the lack of efficacy of this related
peptide, DGAA, at a much higher concentration (up to 1.5 mM). Further
specific questions of sequence specificity were asked and are outlined below.

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Fig. 1.
DGEA activates Ca2+ signaling
pathways in individual Saos-2 cells in a sequence-specific fashion.
Fura 2-loaded cells were treated first with 1.5 mM DGAA and then with
500 µM DGEA, as indicated. Fluorescence ratios for cells were
transformed to Ca2+ concentration
as described in MATERIALS AND METHODS.
Data are Ca2+ levels in 20 cells ± SE.
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Is peptide activity dependent on the negatively charged, acidic
nature of the peptide?
Data in Fig. 2 demonstrate that reducing the net charge
on DGEA by one or two units does not significantly affect the activity of this peptide structure. This was true whether the alteration in
charge was achieved by the addition of the basic amino acid lysine
(KDGEA, net charge
1: this is a continuation of the native sequence found in type I collagen) or by the substitution of the acidic
residues for their uncharged homologues, asparagine or glutamine (NGEA
and NGQA, net charges of
1 and 0, respectively). There was a
reduction in potency with the NGQA peptide. To activate an equivalent
number of cells with NGQA as with 290 µM DGEA, it was necessary to
use 480 µM. These data, coupled with the complete lack of activity in
the DGAA peptide (Fig. 1 and Table 2), lead us to
suspect that the functional activity of the DGEA sequence may center on
the glutamate residue.

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Fig. 2.
Structure-function analysis for DGEA and related peptides. Data are
mean percentage of cells responding to the indicated concentrations of
peptide. For DGEA, n = 56 cells in 10 experiments; for the other peptides, n = 12 cells in 3 experiments. No significant differences between
peptides at particular concentrations were observed (Mann-Whitney's
U test). Peptide sequence, when cells
are activated, has a steep concentration gradient. Minor variants of
the sequence that produce cell activation exhibit similar
characteristics.
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Is the COOH-terminal alanine residue important for activity?
Staatz et al. (17) reported that KDGE failed to inhibit platelet
adhesion to collagen and from this inferred that the COOH-terminal alanine was important for activity. Our data indicate that, in Saos-2
cells at least, KDGE is as effective as KDGEA and DGEA at mobilizing
Ca2+; the removal of the
COOH-terminal alanine from KDGEA (to give KDGE) did not significantly
reduce peptide activity in our system.
Is the spacing of the two acidic amino acids important for activity?
The further separation of the charged residues by inserting additional
glycine or alanine-glycine units (DGGEA and DGAGEA) again had no
significant effect on the Ca2+
mobilizing activity of the peptide. However, there does appear to be a
trend toward reduced activity with additional spacing of amino acids;
thus 320 µM DGGEA was 20% less effective than DGEA at this
concentration, and 320 µM DGAGEA was 38% less effective (Table 2).
This may also point to the "DG" at the beginning of the sequence
of the molecule being functionally the most important (see above), the
activity decreasing as the distance apart of the two subparts of the
collagen peptide was increased.
Is there any functional significance of these variations of peptide
sequence?
We sought to identify a functional event early on in the activation of
cells, that is, the ability of the cell to attach to its substrate.
These studies demonstrated that adhesion to collagen was significantly
blocked by the following peptides: DGEA, KDGEA, NleDGEA, NGQA, and NGEA
(Fig. 3; NGQA acted to partially prevent adhesion). This shows that, despite the modifications of peptide sequence to modify the charge, there was little variation in function. That is, NGQA and NGEA were very similar in activity to DGEA.

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Fig. 3.
Results of the cell adhesion assay with crystal violet (see text for
details). For DGEA and similar peptides, binding to the substrate was
blocked and cells were removed by washing. Thus no cells are detected
adherent to the collagen gel.
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The data for other peptides (Fig. 2) indicate that the changes made to
the amino acid sequence of DGEA have little effect on either the
potency (Fig. 2) or the efficacy (Fig. 3) of the response. The peptides
were used to address specific questions and can be divided into groups
based on these questions.
Table 2 shows the activity of several additional peptide sequences and
the neurotransmitter amino acid glutamate in our system. There are
several points to note. Most importantly, replacing the large,
negatively charged glutamic acid residue with alanine resulted in a
peptide (DGAA, see also Fig. 1) with negligible activity in this
system, even at 1.5 mM. The two norleucine-containing peptides are
controls for the lysine peptides in Fig. 3. Data obtained with these
peptides simply indicate that the addition of an uncharged homologue of
lysine had a similarly small effect on
Ca2+ mobilization as did the
addition of lysine itself. The data for EGDA show that
swapping the positions of the two charged residues does not influence
the ability of the peptide to mobilize
Ca2+.
Overall, the structure-function analysis suggests that the charge of
the peptide is not important for the activation of
Ca2+ signaling (Fig. 2 and Table
1). Two lines of evidence support this conclusion. First, addition of
an NH2-terminal, positively charged lysine (net charge from
2 to
1) did not decrease
the Ca2+ mobilizing activity of
the peptide. Second, substitution of either aspartic acid by asparagine
or both aspartic acid and glutamic acid by asparagine and glutamine,
respectively, also had little effect of the peptide-induced
Ca2+ signal. Thus a peptide with
zero net charge but with nearly identical side chains to DGEA (i.e.,
NGQA) was almost as effective in mobilizing Ca2+ as DGEA itself. The lack of
activity found with DGAA (net charge
1) also suggests that
charge is not the basis of the mechanism of action of DGEA. We believe
that the size and shape of the amino acid side chains are a much more
important factor regulating the activity of this peptide. Finally, free
glutamate and a neutral tetra-alanine peptide had no
Ca2+-mobilizing activity on Saos-2
cells.
We also established a concentration-response curve for DGEA and all
related peptides (Fig. 2). This response for DGEA specifically is
concentration dependent, with an
EC50 of ~250 µM and maximal elevation of Ca2+ produced by
500-700 µM DGEA. The EC50
is approximately one order of magnitude lower than the apparent
dissociation constant for DGEA inhibition of platelet
adhesion to collagen (16).
Extracellular Binding of DGEA
There are many complexities of DGEA binding to Saos-2 cells that
involve additional aspects other than integrin binding. Two specific
aspects we investigated were the dependence on divalent cations and the
relevance of an intact cytoskeleton. Specific questions we examined are
outlined below.
Does binding of DGEA require
Ca2+?
In a previous report, EGTA was used to eliminate
Ca2+ from the extracellular fluid
and thus eliminate the potential for influx of the ion (12). We thought
it likely that the initial component of the signal of DGEA activation
would be derived from intracellular Ca2+ stores. To test this, we
treated cells with DGEA in the absence of external
Ca2+ (cells were rinsed three
times with Ca2+-free
HEPES-buffered salt solution after serum starving for 24 h; the
solution contained 1 mM EGTA and was added 1 min before addition of
DGEA). These cells were unable to demonstrate a rise of
Ca2+ concentration (Fig.
4, n = 20), but the
addition of ryanodine (2 µM) clearly showed release from
intracellular stores.

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Fig. 4.
Data are mean responses ± SE of 10 cells to 2 sequential
treatments, one with 725 µM DGEA and the second with 2 µM
ryanodine, all in the presence of EGTA.
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However, these data were further investigated to establish if
Ca2+ was essential for binding of
the DGEA to the integrin. In these experiments, we used DGEA bound to
FITC. The Ca2+ mobilizing activity
was confirmed, before use as a labeling compound for DGEA with FITC
attached, by adding 500 µM to cells to replicate that activity of
DGEA (data not shown). Cells had to be fixed within 90 s of stimulation
by this variant due to the inhibition of cell adhesion; only limited
numbers of cells were visible in each field, but fluorescent levels
were consistent in each experiment. Cells with DGEA alone showed no
fluorescence (Fig.
5A); cells with
divalent cations (Ca2+,
Ba2+, and
Mg2+) all showed fluorescent
binding of DGEA and FITC (Fig. 5,
B-D); and cells without Ca2+ or with
La3+ replacing
Ca2+ showed no fluorescence (Fig.
5, E and
F). This would imply the need for
divalent cations to exist for binding of the DGEA to the cells. The
La3+ replacing
Ca2+ would have obstructed
Ca2+ channels and should prevent
Ca2+ from being released into the
medium from the cells.

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Fig. 5.
A series of fluorescent photomicrographs showing the binding of DGEA
with Saos-2 cells. In A, DGEA binds but without FITC. No
cells are visible. Divalent ions appear important in the binding
process, as Ca2+
(B),
Ba2+
(C), and
Mg2+
(D) all appear to allow binding.
However, the number of cells is significantly reduced as a consequence
of the DGEA effects on cellular adherence (see Fig. 3). Finally,
removal of Ca2+
(E) and replacement with
La3+
(F) appear to have no fluorescent
cells.
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Does the interaction of DGEA with the cell require an intact
cytoskeleton?
Interactions of the cell substrate are often reported as requiring an
intact actin cytoskeleton. We have been able to shown from the data in
Fig. 6 that, by blocking the polymerization of actin and
thus eliminating the cytoskeleton, we still are able to increase the
intracellular Ca2+ levels.
Examination of the cells by fluorescence microscopy (data not shown)
clearly supports the blockade of actin polymerization by cytochalasin B
with no visible fluorescence present. However, integrin-specific
interactions have been reported to require an intact actin cytoskeleton
elsewhere, and our work clearly contradicts this observation (20). In
particular, postoccupancy events of integrins are reported as reliant
on this intact cytoskeleton, and failure of this mechanism may well
prevent further cellular activation (21). This does not appear to be
the case in Saos-2 cells.

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Fig. 6.
Cells were loaded and imaged as described in MATERIALS
AND METHODS, except that they were treated with 3 µg/ml cytochalasin B (in 0.3% DMSO) or DMSO alone, for 40 min before
stimulation with 725 µM DGEA. Data are the mean response of all cells
in a field and are representative of 8 other experiments.
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Membrane-Bound
Ca2+ Channels
With the paradoxical lack of activity following activation with DGEA in
the presence of EGTA, we decided to examine the potential role of
extracellular Ca2+ by two
experimental conditions. First, we used
La3+, a known
Ca2+-blocking agent. Second, we
examined the distribution of
45Ca2+
under specific experimental conditions.
The additional experiments revealed that most, if not all, of the
DGEA-induced Ca2+ signal is in
fact derived from intracellular stores. First, 0.5 mM
La3+, the trivalent cation blocker
of receptor-mediated Ca2+ influx
(22), had no effect on the Ca2+
signal generated following DGEA treatment (Fig. 7,
n = 10 cells). Ca2+ for this response must,
therefore, be derived from internal stores.

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Fig. 7.
Two applications of 500 µM DGEA are placed on cells, with second
application in the presence of 500 µM
LaCl3
(n = 10, ±SE).
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Further experiments using 45Ca
supported the use of La3+ as an
inorganic blocker of Ca2+ channels
and prevented entry of Ca2+ into
the cells as a consequence of DGEA activity (Fig. 8).
This was clearly demonstrated by the failure to detect any
radioactivity in the cells following stimulus with DGEA and blockade
with La3+ unless the
45Ca2+
had been included in the medium in which the cells had been cultured for 3 days before the experiment (groups 1 and
2). Even when thrombin stimulation
was used, La3+ obstructed any
increase in intracellular levels from extracellular sources. This was
confirmed by labeled Ca2+ and
imaging studies (data not shown).

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Fig. 8.
To confirm that La3+ obstructed
Ca2+ channels following DGEA
activity, 45Ca was assessed within
the cells. Radioactivity was assessed following stimulation and
normalized as a percentage of the value of activity in unstimulated
cells grown in the presence of 3 µCi of
45Ca2+
(n = 5 Labtek chambers). In the first
series of experiments, thrombin is clearly seen increasing the levels
of intracellular
45Ca2+
but is obstructed by La3+
(A). DGEA increases
45Ca2+
content but not to same level as thrombin.
La3+ does not appear to decrease
the response (B).
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Physiology of Internal Stores: Ryanodine and U-37122
DGEA was also unable to elevate intracellular
Ca2+ levels after preincubating
the cells with 2 µM ryanodine, which is known to empty intracellular
Ca2+ stores. The addition of 10 µM ryanodine obstructs the passage of
Ca2+ out of these stores (3) (Fig.
9, A and
B). The application of DGEA to
ryanodine-treated cells provided evidence to suggest that DGEA promotes
a release of Ca2+ from
intracellular stores and is responsible for the total magnitude of the
signal, none apparently coming from the exterior source.

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Fig. 9.
A: intracellular stores were emptied
with 2 µM ryanodine, and then cells were loaded with DGEA before
there was a possibility of the stores reloading with
Ca2+
(n = 10).
B: sufficient ryanodine was used to
block the diffusion of Ca2+ out of
the stores (10 µM) (n = 10).
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Further experiments were performed with U-73122, the known
phospholipase C inhibitor, and thrombin, a known G protein activator that releases inositol trisphosphate
(IP3) and thus
IP3-sensitive Ca2+ stores (19). In these
experiments, initially without U-73122, DGEA and thrombin responses
were not blocked (n = 10, Fig.
10A); two distinct
peaks of Ca2+ activity were seen.
Addition of U-73122 (10 µM) eliminated the response to thrombin but
essentially allowed a similar response to DGEA (Fig.
10B). These data demonstrate that
the Ca2+ signal generated by
treatment of cells with DGEA is unequivocally derived from
intracellular stores, an observation that argues strongly for
receptor-mediated transduction of the DGEA signal. In addition, the
data support the existence of two distinct pools of
Ca2+ capable of release by two
diverse signaling pathways.

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Fig. 10.
Demonstration that the use of an inositol trisphosphate
(IP3)-mediated agonist failed to
prevent DGEA activity, implying 2 types of functional pools. Thrombin
was added at 0.3 mU/ml to cells followed by 500 µM DGEA.
A: 2 distinct peaks are seen in first
series of experiments. B: inclusion of
U-73122 abolishes the thrombin response.
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Table 2 summarizes Ca2+ response
data for a variety of peptides related to DGEA. A characteristic of all
the peptides was that the induction of the
Ca2+ signal was essentially an
all-or-nothing event; thus, at lower concentrations, a smaller
proportion of cells still give a strong Ca2+ signal.
To summarize our findings, we have described a
Ca2+ mobilizing activity
associated with the DGEA collagen ligand motif that shows partial
sequence specificity. Furthermore, the DGEA peptide activates release
of Ca2+ only from internal stores,
indicating that a specific transmembrane signal transduction event is
being activated. Furthermore, we demonstrate that the activity of the
peptide sequence is not simply related to its highly acidic nature but
that it depends on the structural characteristics of the amino acid
side chains.
Collagen is the major component of the organic matrix in bone and as
such it is likely that it has profound effects on the recruitment and
function of osteoblasts. Data are accumulating from studies in rodent
models of osteoblast function that this is indeed the case (2, 10, 18).
The role of collagen in modulating the function of human osteoblasts is
less well described, but its potential importance is supported by the
data on collagen peptide (DGEA)-induced intracellular
Ca2+ mobilization presented here.
Peptides based on the DGEA collagen motif may prove useful tools in the
analysis of the interaction of human osteoblasts with the organic
matrix of bone and in other systems. In addition, the data suggest that
the activation of a ubiquitous intracellular signal transduction
pathway may be central to the regulation of osteoblast function by the
bone matrix.
 |
ACKNOWLEDGEMENTS |
We wish to thank Drs. H. Aptel and N. Freestone for their comments on
the manuscript and A. Northrop for synthesizing the peptides.
 |
FOOTNOTES |
Address for reprint requests: F. McDonald, Bone Biology Unit, Dept. of
Orthodontics and Paediatric Dentistry, Floor 22, Guy's Tower, St.
Thomas St., London SE1 9RT, UK.
Received 18 June 1997; accepted in final form 9 March 1998.
 |
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