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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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 alpha 1-3,5 and beta 1. Indeed, Dedhar (2) provided data suggesting that integrins of the beta 1-family may play a role in the induction of alkaline phosphatase expression by interleukin-1 in human MG-63 osteosarcoma cells. The alpha 1beta 1- and alpha 2beta 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 alpha 1beta 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 alpha 2beta 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 alpha 2beta 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Fura 2-AM, cytochalasin B, U-73122, and ryanodine were from Calbiochem (Nottingham, UK); LaCl3, collagen, human alpha -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.

                              
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Table 1.   Arrangement of treatment groups

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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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.

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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Table 2.   Ca2+ mobilizing activity of control peptides and glutamate on Saos-2 cells

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.

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.

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.

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.

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).

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).

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).

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.

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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Am J Physiol Cell Physiol 275(1):C33-C41
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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