1Medical Research Service, Kansas City Department of Veterans Affairs Medical Center, and 2Department of Biochemistry and Molecular Biology, 3Department of Physical Therapy Education and Rehabilitation, and 4Department of Anatomy and Cell Biology University of Kansas Medical Center, Kansas City, Missouri 64128; and 5Division of Cellular Pharmacology, Department of Signal Transduction, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan
Submitted 1 July 2003 ; accepted in final form 21 March 2004
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ABSTRACT |
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osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate
Numerous publications provide in vivo demonstration that CGRP innervation is associated with bone formation (and similar mineralizing processes), particularly during development, growth, or repair (for detailed review, see Refs. 16 and 17). In vitro studies have demonstrated that CGRP stimulates the osteoblastic differentiation of bone marrow mesenchymal stromal cells (32, 33) and acts directly on osteoblastic cells to modulate phenotypic functions (7, 29, 37). These and similar data suggested that neuroactive CGRP plays a role in stimulating or maintaining bone formation in skeletal tissues. In further support of the bone anabolic action of CGRP, Vignery and coworkers demonstrated that CGRP protects against ovariectomy-induced bone loss (36) and that targeted transgenic expression of CGRP in mouse osteoblasts increases bone density 2740% at skeletal sites by stimulating cancellous bone formation (1).
Despite data suggesting that CGRP can stimulate bone formation, its mechanism of action in osteoblast-related cells is not well understood. Historically, attention was drawn to CGRP's pronounced stimulation of cAMP formation in immature osteoblast-related cells isolated from bone and bone linings from several species (28), but this cAMP response varies considerably in different osteoblastic cell types (Refs. 3, 19, and 28; Burns and Kawase, unpublished observations). In addition, we (18, 20, 21) and others (11, 12, 38, 39) have recently reported that CGRP can stimulate cAMP-independent intracellular signaling pathways in several osteoblastic cell types. We thought it significant that mature osteoblast-like cells respond to CGRP by increasing intracellular Ca2+ ([Ca2+]int) and/or altering membrane potential (Em) while yielding small increases (if any) in cAMP production (18, 20, 21). Similarly, Drissi et al. (11, 12) demonstrated that CGRP increases [Ca2+]int, but not cAMP, in human OHS-4 osteoblast-like cells. Recently, Villa et al. (38, 39) demonstrated that CGRP's 2.2-fold stimulation of proliferation in primary cultures of human osteoblasts is cAMP independent. Collectively, these data suggest that cAMP may not represent the primary mode of CGRP signaling in mature osteoblastic cells.
From among an assortment of osteoblast-like cell lines, clonal osteosarcoma-derived human MG-63 cells have been described as a model for early-stage immature preosteoblasts because of the nature of their response to factors like l,25(OH)2-vitamin D3 and their regulated temporal development of bone-type alkaline phosphatase activity (4, 9). We very recently found (22) that MG-63 cells prominently express the type-1 CGRP receptor [calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)1] and respond to CGRP with 35-fold increases in cAMP formation and rapid phosphorylation or dephosphorylation of several types of mitogen-activated protein kinase. Similar strong cAMP signaling has also been reported for immature lining and preosteoblastic cells (3, 19, 28, 34), and this correlation suggests that cAMP and cAMP-dependent pathways could be the primary mode of CGRP signaling in immature osteogenic cells.
Thus it was of considerable interest to determine whether CGRP also induces changes in [Ca2+]int or cellular Em in preosteoblastic human MG-63 cells. To this end, we have employed a sensitive, single whole cell fluorescent confocal analysis using a fluo 4-AM-Fura red-AM ratiometric assay to assay [Ca2+]int and similarly modified our bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)] bis-oxonol assay to monitor Em. Expecting to find modest or negligible effects of CGRP on these parameters, we have instead demonstrated robust effects that exceed in complexity and magnitude those we had previously observed in a more mature model of osteogenic cells (18, 20, 21). In addition, we also report unexpected differences between our present results and our previous findings, including CGRP's activation of cAMP-dependent, verapamil-sensitive membrane Ca2+ channels to produce a long-sustained Ca2+ increase in MG-63 cells. Although cAMP-independent mechanisms figure into induction of the initial Ca2+ transient and polarization of Em as previously observed in UMR106 cells, the situation is substantially more complex in preosteoblastic MG-63 cells and could conceivably involve additional CGRP receptor subtypes.
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MATERIALS AND METHODS |
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Cells and cell cultures. Low-passage MG-63 osteogenic human osteosarcoma cells were obtained from the American Type Culture Collection (Manassas, VA) and plated, grown, and maintained in DMEM supplemented with 1 mM L-glutamine, standard penicillin-streptomycin, and 2% fetal bovine serum (FBS; JRH Biosciences, Mission, KS). Cells were normally plated at a density of 1 x 103 cells/cm2 and cultivated in humidified 5% CO2-95% air at 37°C. Cultures were never allowed to become confluent. For the assays described below, cells were grown on sterile 1-mm-thick, 23-mm-diameter circular coverslips. All assays were performed in physiological saline solution (PSS; in mM: 130 NaCl, 5 KCl, 1.5 CaCl2, 1 glucose, 10 HEPES, pH 7.30).
Measurement of [Ca2+]int. The data reported here were obtained with detailed single-cell measurements of [Ca2+]int using the dual-dye ratiometric method of Lipp and Niggli (25, 26) and laser-scanning fluorescent confocal microscopy. In this protocol, subconfluent densities of MG-63 cells grown on circular 1-mm-thick coverslips were loaded for 30 min with 1 µM fluo 4-AM and 4 µM fura red-AM in standard DMEM supplemented with 1% FBS. Slides were rinsed twice with PSS + 0.2% FBS, mounted individually to a 23-mm-diameter Attafluor slide chamber (Molecular Probes), immersed in 1 ml of PSS + 0.2% FBS, and then examined at 22°C on the stage of an inverted Nikon Eclipse T-300 microscope equipped for fluorescent dark-field imaging and coupled directly to a Bio-Rad RadiancePlus 2000 triple-laser computer-controlled scanning confocal apparatus (Bio-Rad Laboratories, Hercules, CA).Experimental treatments were introduced by withdrawing 400 µl of the chamber bath volume, into which was mixed the desired treatment (peptide or reagent). This solution was carefully introduced back into the slide-holding chamber with careful mixing, thus avoiding exposure of cells to unusually high levels of vehicle or treatment reagent.
To estimate [Ca2+], dye-loaded cells were excited with the 488-nm line of an argon laser running at approximately <1.5 mW. The standard confocal pinhole setting for this assay was 3.2 mm, and when emissions were collected with the x40 or x60 objectives the "optical slice" was typically 0.6 µm thick. Fluo 4 emission at 520 ± 15 nm strongly increases as [Ca2+] increases, whereas fura red emission at 620 ± 40 nm decreases as [Ca2+] increases. These two emissions from individual mouse-delineated cellular cross sections were collected in separate channels. General cellular fluorescence was found to be so low as to be negligible in these confocal assays, but backgrounds were still collected in each assay.
Time course profiles were initially collected as raw data (total pixels within a computer-designated cellular cross section for each designated emission), and a background time course (equivalent areas without cells, or identical cells without dye) was collected and stored for each assay and subtracted from each time course emission. Ratioing of these two fluorescent emissions provides a fluorescent report of [Ca2+] levels that is independent of dye-loading patterns and similar problems. Evaluating this ratio vs. a [Ca2+] standard curve prepared in cell homogenates and read on a coverslip with the confocal microscope under identical conditions (pinhole, laser power, objective, emission settings) permitted accurate estimation of [Ca2+]int. These values generally agreed with values obtained through identical assays conducted instead with fura 2 and a UV laser-equipped Zeiss confocal microscope (Burns and Stehno-Bittel, unpublished data) and are presented as a quantitative estimation of [Ca2+]int.
Although these assays require additional steps in data processing, the mean population analysis of a field of single cells yields a result that is identical, albeit with better signal-to-noise ratio, to what is measured in a cuvette assay (data not shown). These [Ca2+] assays were most reliable when conducted in PSS + 0.2% FBS, because most cells displayed periodic 1020% fluctuation in [Ca2+]int when studied in DMEM + 0.2% FBS (possibly because of the presence of the ionotropic glutamate receptor channel).
Measurement of Em. The basic method for measuring changes in cellular Em was similar to what we have previously reported (18) and is a modification of Civitelli's original protocol (8). Subconfluent MG-63 cells grown on coverslips were rinsed with PSS containing 0.2% FBS and then incubated for 40 min in the same medium supplemented with 0.5 µM levels of the bis-oxonol dye DiBAC4(3). As cells polarize, the 500- to 600-nm fluorescent emission from DiBAC4(3) decreases (5, 8), and pilot experiments conducted in a quartz cuvette with a Shimadzu RC-5391-PC spectrofluorometer confirmed this phenomenon.
The data reported here were generated by single whole cell assay of Em changes in MG-63 cells plated onto circular 1-mm coverslips with laser-scanning confocal fluorescent microscopy (as above). Excitation was with the 488-nm line of the argon laser (running at 1.5 mW), and the 500- to 620-nm fluorescent emission (620-nm dichroic mirror + 500-nm LP emission filter) was collected. Bis-oxonol dye is not as bright as fluo 4, so a 3- to 3.5-mm pinhole was used, yielding an effective optical slice of 0.8 µm when the x40 or x60 objectives were used. These data were collected to permit estimation of relative Em values vs. that of an initial 5-min baseline (see below). Between 8 and 20 cells were studied in each microscopic field. As before, data were collected and stored as time course profiles.
Data analysis. For both assays the specific cellular fluorescence (defined as the total number of bright pixels/cell cross section background intensity pixel data) was imported into MicroSoft Excel. Accurate [Ca2+] values were obtained as described above. For Em time courses, each data string from a cell was normalized vs. the initial average baseline (average initial fluorescent pixel count/cell over 5 min) to adjust for differences in the area of cellular footprint. It has so far proven impossible to quantitate the DiBAC4(3) emission against a standard curve to produce quantitative estimation of Em values; thus all Em data are presented as fluorescence intensity of emission as a fraction of the initial value, much as we did previously (18).
Pooling of single-cell recordings and aggregate analysis. It was important to determine the overall response within a given microscopic field, even if individual cells were heterogeneous in their time course response. Thus the "mean population value" for each time point was determined by averaging the aggregate value from each of the cells being examined in a particular experiment, and this mean value ± SE is plotted for each time point. In this manner, rather complex responses could be analyzed for general parameters and overall properties. It is of interest that the aggregate response of a number of imaged cells (generally when n > 6 cells) strongly resembles the data obtained from analyzing hundreds of adherent cells in cuvette-sized assays in the spectrofluorometer. Whenever the magnitude of a response with one treatment was evaluated against the magnitude obtained with a different treatment, the area under the curve (AUC) for the corresponding time interval from each result was estimated and directly compared.
Statistical analysis. Time course profiles that are plots of the mean population value determined by averaging the same time point from the individual profiles recorded in 822 individual cells display means ± SE. For every case, the standard deviation is <5% of the mean value shown. When necessary, mean population values are compared point by point at areas of interest against the basal mean population profile using a simple two-tailed Student's t-test to determine whether the values are different with a statistical significance of P < 0.05 or better. With regard to multiple comparisons and a single set of controls, comparisons were by one-way ANOVA with group comparisons made by Tukey's test. Again, P < 0.05 was accepted as significant.
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RESULTS |
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Figure 1 demonstrates CGRP-induced increases in [Ca2+]int in >90% of MG-63 cells within 45 s of application of 10 nM human CGRP (Fig. 1, B vs. A). Additionally, >80% of responsive cells increased [Ca2+] in nuclear and/or perinuclear regions as part of this response. Neither salmon nor human calcitonin at doses of 1100 nM had any significant effect on [Ca2+]int, and no effects were observed with equal volumes of 0.01% trifluoroacetic acid or 0.1% DMSO (as vehicle controls). Baseline recordings of vehicle-stimulated individual cells revealed only minor 12% fluctuations in fluo-4-to-fura red ratio, and simultaneous imaging of fluo 4 demonstrated little falloff in signal intensity over 1020 min of laser scanning.
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Averaged time course changes in CGRP-increased [Ca2+]int in single attached MG-63 cells. As suggested by multiple single-cell profiles, >90% of MG-63 cells responded to 10 nM CGRP with an obvious two-phase change in [Ca2+]int. Overall similarities were analyzed in more detail by collecting 1122 single-cell profiles in an individual experiment and determining the mean population value at each time point ± SD. Three different examples of summed aggregate data for MG-63 cells stimulated with 10 nM human CGRP are presented in Fig. 2 (A and B demonstrate two separate experiments containing 16 or 22 cells, respectively). As summarized in Fig. 2C, analysis of 270 single adherent MG-63 cells in 15 additional experiments demonstrated essentially the same biphasic time course.
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Averaged dose-response relationship in CGRP-increased [Ca2+]int in single attached MG-63 cells.
As shown in Fig. 3, the area under the initial peak of CGRP-stimulated [Ca2+]int was calculated and plotted against CGRP dose to determine dose response. A mean population value profile was determined for each concentration of CGRP (as before), and a number of experiments with 0.0180 nM human CGRP demonstrated a dose-response curve. Maximal effects were obtained by 20 nM, and both 40 and 80 nM CGRP produced substantially less of an increase than 20 nM. Increases were statistically significant at P < 0.05 (or better) for doses of 0.01 nM CGRP and greater. The apparent EC50 was
0.25 nM CGRP. The secondary phase [Ca2+]int response was also analyzed separately and displayed a very similar EC50 (data not shown).
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Using DiBAC4(3) bis-oxonol dye and confocal microscopy, we next monitored the relative cellular Em of single adherent MG-63 cells treated with 10 nM human CGRP (Fig. 6). More than 90% of CGRP-stimulated MG-63 cells responded within 50 s and lost 20% of their bis-oxonol fluorescence over 300 s (Fig. 6, B vs. A). Because bis-oxonol fluorescent emission decreases in parallel with increased negative polarity of cellular Em, these large changes represent significant hyperpolarization of the membrane potential. Unfortunately, it was not possible to quantify the signal from this dye, but DiBAC4(3) emission has been reported to decline
25% with a 100-mV shift in Em (5). Parallel experiments in which vehicle (0.01% trifluoroacetic acid) alone was added demonstrated that no more than a 23% decline in DiBAC4(3) fluorescent emission normally occurs over 900 s of low-level laser illumination.
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Averaged time course and dose-response relationship of CGRP-induced Em hyperpolarization in adherent MG-63 cells.
When all single-cell records from independent experiments (with 720 cells) were normalized vs. original baseline emission intensities over 5 min (to correct for cellular "footprint"see MATERIALS AND METHODS) and used to calculate mean population values for each time point, the resulting plot demonstrated the general similarity in the averaged response of the cell population (Fig. 7). Data from an initial experiment (Fig. 7A) and summary data from 15 individual experiments examining a total of 180 cells (Fig. 7B) in which MG-63 cells were stimulated with 20 nM human CGRP proved to be very similar. The vast majority of cells responded by 50 s to 20 nM human CGRP with a steep initial drop in bis-oxonol fluorescence, a partial reversal, and then a second steep drop to yield maximal reduction in fluorescence after 300 s. Hyperpolarization of Em was sustained for at least 600 s.
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Characterization of CGRP-induced Em hyperpolarization.
A number of multicell experiments were performed using the maximally effective dose of CGRP (20 nM) with standard concentrations of specific or selective inhibitors to determine the involvement of different pathways or membrane channels in CGRP-induced hyperpolarization of Em (Fig. 8). Rp-cAMPS (30 µM; 20-min pretreatment) produced an 8% reduction in effect 300 s after treatment that was not statistically significant; however, it did significantly delay the onset of Em polarization, possibly by eliminating the initial phase of polarization. A selective inhibitor of Ca2+-activated K+ (KCa) channels, charybdotoxin (5 µg/ml; 5-min pretreatment), attenuated CGRP-induced reduction in bis-oxonol emission by 20% (with P < 0.05 vs. CGRP alone), whereas a specific inhibitor of ATP-sensitive K+ (KATP) channels, glyburide (1 µM; 5-min pretreatment), blocked it by 83% (P < 0.001).
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DISCUSSION |
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In investigating this concept, we previously studied the effects of CGRP on [Ca2+]int and Em in mature osteoblastic rat UMR106 (18, 20, 21) and RCOB-3 (20) cells. CGRP did not elicit large increases in cAMP production (Kawase and Burns, unpublished observations), whereas it did invoke sizable Ca2+ transients and Em hyperpolarization in UMR106 (18, 20, 21) and RCOB-3 cells (Ref. 20; Ca2+ data not published). This is consistent with a subsequent demonstration (6) that UMR106 cells do not seem to express the type 1 CGRP receptor (which generally couples tightly to adenylate cyclase activation). Thus these findings and demonstration of similar strong cAMP-independent CGRP signaling in other mature osteoblastic cell lines (11, 12) and primary human osteoblasts (38, 39) strongly suggested that cAMP-dependent signaling pathways are no longer the predominant mediators of CGRP action in mature osteoblastic cells (18). On the basis of previous data (3, 28, 34), the converse argument could also be madethat cAMP-dependent signaling pathways predominate in immature osteogenic cells.
We have now tested in preosteoblastic human MG-63 cells the working hypothesis that cAMP-dependent mechanisms would predominate without appreciable contribution from cAMP-independent signaling pathways. MG-63 cells were previously shown to strongly express mRNA for one of the components (CRLR) of a type 1 CGRP receptor (35). We recently demonstrated (22) the expression of both CRLR and RAMP1 proteins in these cells and determined that CGRP strongly stimulates cAMP formation. We could not detect expression of multiple CGRP receptors by analyzing saturation-binding kinetics with 125I-His10-labeled human CGRP during the course of these studies (22). On the basis of these data, we assumed that only one receptor subtype was present and that it would predominantly signal through cAMP-dependent pathways. Thus we investigated [Ca2+]int and Em values during CGRP treatment just to verify that no changes occurred. As shown in the present study, CGRP produces strong changes in both these parameters. The results reported here show both unexpected similarities to and surprising differences from our data from UMR106 cells, and, on evaluation, these data require that we discard or reformulate our simple hypothesis.
With regard to methodology, we have in the present study introduced and successfully adapted the dual-dye confocal fluorescent microscopy method of Lipp and Niggli (25, 26) to assay [Ca2+]int within single human preosteoblastic MG-63 cells. Applying similar protocols has also greatly improved our established bis-oxonol assay of relative cellular Em. To simplify these complex actions, we have only considered individual whole cells; future studies will need to examine apparent subcellular changes visualized by fluorescent imaging within these cells. We have now measured large and complex CGRP-induced changes in [Ca2+]int and cellular Em that were not predicted from our working hypothesis.
CGRP-induced two-phase changes in [Ca2+]int over time and mechanism of action. In human osteoblastic OSH-4 cells, CGRP was shown to induce a 2.3-fold initial peak in [Ca2+]int that lasts for >30 s before subsiding into a sustained plateau maintained at 140% of basal levels for an additional >60 s (11, 12). Such a response to CGRP had not previously been shown, because in our studies of rat osteoblastic UMR106 cells, CGRP elicited only simple 30- to 45-s Ca2+ transient peaks that quickly subsided (20). In the present study, the Ca2+ increase in CGRP-treated MG-63 cells featured both a large initial transient and a subsequent sustained phase. This two-phase profile itself is similar to that obtained from CGRP-treated OHS-4 cells and is very similar to that induced by parathyroid hormone (PTH) in UMR106 cells (43, 44).
The initial transient peak was inhibited 75% by thapsigargin but not substantially affected by extracellular EGTA or Rp-cAMPS, indicating that this response is primarily produced by Ca2+ release from intracellular stores in a cAMP-independent manner. However, because thapsigargin showed an incomplete elimination of this phase and both EGTA and verapamil produced a small inhibition, there may be a small contribution to the initial phase (perhaps 1525% of the total effect) through very rapid transmembrane Ca2+ influx as has been shown for PTH (44).
In contrast, the second sustained phase of elevated [Ca2+]int in MG-63 cells was not appreciably sensitive to thapsigargin but was inhibited by extracellular EGTA and verapamil. This phase was also inhibited by Rp-cAMPS and compound H-89 (data not shown) and eliminated by 2 µM CGRP(837). These data suggest that, in the second phase, activated CGRP receptors stimulate Ca2+ influx through a voltage-dependent Ca2+ channel and this process is initiated by cAMP and further integrated by cAMP signaling pathways. Similarly, in PTH-treated UMR106 cells, the slow secondary phase was demonstrated to be mediated by a cAMP-dependent, verapamil-sensitive membrane Ca2+ channel (44). In addition, the secondary phase observed in CGRP-treated OHS-4 cells, which is substantially smaller than that obtained here, was also shown to result from transmembrane influx as it was nifedipine sensitive; however, the secondary phase in OHS-4 cells is cAMP independent (11, 12).
CGRP-induced membrane hyperpolarization in MG-63 cells. Compared with Ca2+ mobilization, CGRP action on Em has rarely been studied in osteoblastic cells at any stage of differentiation. In our previous study (18), we for the first time demonstrated with standard cuvette assays that CGRP induces membrane hyperpolarization in adherent UMR106 cells. Judging from the dose-response relationship and the very small cAMP response elicited by CGRP in these cells, we suggested that cAMP-dependent signaling pathways were not involved in this action. In the present study of human preosteoblastic MG-63 cells using more sensitive protocols, we were surprised to find very similar time course profiles and dose-response relationships.
This action was further characterized with several specific pharmacological inhibitors. Rp-cAMPS delayed or suppressed the most initial transient phase of Em polarization but failed to significantly inhibit the sustained phase. Conversely, glyburide almost completely blocked the second phase but not the initial phase, whereas charybdotoxin showed only slight effects on either phase. Therefore, these findings suggest that the initial phase is induced by a cAMP-dependent mechanism (possibly the rapid opening and closing of a Cl channel) and the large sustained major phase is produced by activating cAMP-independent KATP channels. However, our experiments do not rule out the possibility that either phase is also somewhat influenced by a Ca2+-dependent mechanism.
Hints of a second CGRP receptor subtype. CGRP(837) has been described as a selective antagonist of type 1 CGRP receptors. CGRP(837) was able to eliminate certain effects (secondary phase of increased [Ca2+]int; Fig. 5C) while only attenuating other effects by 55% [initial Ca2+ peak (Fig. 5C) and the bulk of cellular Em polarization (Fig. 8)]. Although modest, the differences in level of inhibition between the two sets of effects did suggest that different receptors could be involved in this process. Unfortunately, careful analysis of the recent literature reveals several reports (40, 42) questioning how selective CGRP(837) actually proves to be (31). Because these pharmacological differences are the major reason to propose the existence of multiple subtypes of CGRP receptor (31), this ambiguity has led Abel and coworkers (40) to propose that type 2 receptors essentially represent a pharmacological illusion reflecting receptor number and the efficiency with which receptor is coupled to the intracellular transduction machinery. Considerably more work will be required to generate definitive data that determine whether a type 2 receptor is expressed on MG-63 cells.
Proposed explanation and scheme. Our best interpretation of the data described and discussed here is summarized in Fig. 9, which indicates the different signaling pathways and effector molecules used by CGRP in MG-63 cells. The simplest explanation is that a type 1 CGRP receptor concomitantly couples to two or more different effector pathways in MG-63 cells. What would once have seemed an unlikely explanation is now supported by the literature, which indicates that transfected CGRP receptors can concomitantly signal by increasing both cAMP and Ca2+ (24, 31). Dual signaling has also been shown for the primary calcitonin receptor (31). Typically, changes in Em have not been followed, and the exact mechanism(s) by which cellular Em is modulated in osteogenic cells remains to be identified.
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These data further support the basic concept that bone is an important target tissue for the peripheral nervous system (13, 14, 16, 17, 27). Combined with our previous data (18, 2022), these data provide a number of notable findings that collectively improve our understanding of CGRP-activated signaling pathways in osteoblastic cells.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bean AJ, Zhang X, and Hokfelt T. Peptide secretion: what do we know? FASEB J 8: 630638, 1994.
3. Bjurholm A, Kreicbergs A, Schultzberg M, and Lerner UH. Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and SaoS-2) and primary bone cells. J Bone Miner Res 7: 10111019, 1992.[ISI][Medline]
4. Bonewald LF, Kester MB, Schwartz Z, Swain LD, Khare A, Johnson TL, Leach RJ, and Boyan BD. Effects of combined transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63). J Biol Chem 267: 89438949, 1992.
5. Brauner T, Hulser DF, and Strasser RJ. Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochim Biophys Acta 771: 208216, 1984.[ISI][Medline]
6. Buhlmann N, Leuthauser K, Muff R, Fischer JA, and Born W. A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. Endocrinology 140: 28832890, 1999.
7. Burns DM and Kawase T. Calcitonin gene-related peptide, amylin, or parathyroid hormone stimulate in vitro biomineralization (Abstract). J Bone Miner Res 12, Suppl 1: F392, 1997.
8. Civitelli R, Reid IR, Halstead LR, Avioli LV, and Hruska KA. Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes. J Cell Physiol 131: 434441, 1987.[ISI][Medline]
9. Dean DD, Schwartz Z, Bonewald L, Muniz OE, Morales S, Gomez R, Brooks BP, Qiao M, Howell DS, and Boyan BD. Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of -glycerophosphate and ascorbic acid. Calcif Tissue Int 54: 399408, 1994.[ISI][Medline]
10. Diez-Guerra FJ, Sirinathsinghji DJ, and Emson PC. Evidence for release of calcitonin gene-related peptide-like immunoreactivity from nerve fibers containing substance P-, somatostatin-, and vasoactive intestinal polypeptide-like immunoreactivity. Histochemistry 91: 3538, 1988.[ISI]
11. Drissi H, Lasmoles F, Le Mellay V, Marie PJ, and Lieberherr M. Activation of phospholipase C-1 via G
q/11 during calcium mobilization by calcitonin gene-related peptide. J Biol Chem 273: 2016820174, 1998.
12. Drissi H, Lieberherr M, Hott M, Marie PJ, and Lasmoles F. Calcitonin gene-related peptide (CGRP) increases intracellular free Ca2+ concentrations but not cyclic AMP formation in CGRP receptor-positive osteosarcoma cells (OHS-4). Cytokine 11: 100107, 1999.
13. Garcia-Castellano JM, Diaz-Herrera P, and Morcuende JA. Is bone a target-tissue for the nervous system? New advances on the understanding of their interactions. Iowa Orthop J 20: 4958, 2000.[Medline]
14. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24: 739768, 1988.[CrossRef][ISI][Medline]
15. Hosoi J, Murphy GF, Egan CL, Lerner EA, Grabbe S, Asahina A, and Granstein RD. Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363: 159163, 1993.[CrossRef][ISI][Medline]
16. Imai S and Matsusue Y. Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone. Microsc Res Tech 58: 6169, 2002.[CrossRef][ISI][Medline]
17. Irie K, Hara-Irie F, Ozawa H, and Yajima T. Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech 58: 8590, 2002.[CrossRef][ISI][Medline]
18. Kawase T and Burns DM. Calcitonin gene-related peptide stimulates potassium efflux through adenosine triphosphate-sensitive potassium channels and produces membrane hyperpolarization in osteoblastic UMR106 cells. Endocrinology 139: 34923502, 1998.
19. Kawase T, Howard GA, Roos BA, and Burns DM. Calcitonin gene-related peptide and parathyroid hormone acutely block calcium-uptake in osteoblastic cells by different mechanisms (Abstract). J Bone Miner Res 7, Suppl 1: S207, 1992.
20. Kawase T, Howard GA, Roos BA, and Burns DM. Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentration in rat UMR 106 osteoblastic cells. Bone 16, Suppl 4: 379S384S, 1995.[CrossRef][Medline]
21. Kawase T, Howard GA, Roos BA, and Burns DM. Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels. Endocrinology 137: 984990, 1996.[Abstract]
22. Kawase T, Okuda K, and Burns DM. Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism. Eur J Pharmacol 470: 125137, 2003.[CrossRef][ISI][Medline]
23. Kelly RB. Storage and release of neurotransmitters. Cell 72: 4353, 1993.[ISI][Medline]
24. Kuwasako K, Cao YN, Nagoshi Y, Tsuruda T, Kitamura K, and Eto T. Characterization of the human calcitonin gene-related peptide receptor subtypes associated with receptor activity-modifying proteins. Mol Pharmacol 65: 207213, 2004.
25. Lipp P and Niggli E. Ratiometric confocal Ca2+-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14: 359372, 1993.[ISI][Medline]
26. Lipp P and Niggli E. Submicroscopic calcium signals as fundamental events of excitation-contraction coupling in guinea-pig cardiac myocytes. J Physiol 492: 3138, 1996.[Abstract]
27. Lundberg P, Bostrom I, Mukohyama H, Bjurholm A, Smans K, and Lerner UH. Neuro-hormonal control of bone metabolism: vasoactive intestinal peptide stimulates alkaline phosphatase activity and mRNA expression in mouse calvarial osteoblasts as well as calcium accumulation mineralized bone nodules. Regul Pept 85: 4758, 1999.[CrossRef][ISI][Medline]
28. Michelangeli VP, Fletcher AE, Allan EH, Nicholson GC, and Martin TJ. Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells. J Bone Miner Res 4: 269272, 1989.[ISI][Medline]
29. Millet I and Vignery A. The neuropeptide calcitonin gene-related peptide inhibits TNF- but poorly induces IL-6 production by fetal rat osteoblasts. Cytokine 9: 9991007, 1997.[CrossRef][ISI][Medline]
30. Nelson MT, Huang Y, Brayden JE, Hescheler J, and Standen NB. Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels. Nature 344: 770773, 1990.[CrossRef][ISI][Medline]
31. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, and Foord SM. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 54: 233246, 2002.
32. Shih C and Bernard GW. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides 11: 625632, 1990.[CrossRef][ISI][Medline]
33. Shih C and Bernard GW. Calcitonin gene related peptide enhances bone colony development in vitro. Clin Orthop 334: 335344, 1997.[Medline]
34. Thiebaud D, Akatsu T, Yamashita T, Suda T, Noda T, Martin RE, Fletcher AE, and Martin TJ. Structure-activity relationships in calcitonin gene-related peptide: cyclic AMP response in a preosteoblast cell line (KS-4). J Bone Miner Res 6: 11371142, 1991.[ISI][Medline]
35. Togari A, Arai M, Mizutani S, Mizutani S, Koshihara Y, and Nagatsu T. Expression of mRNAs for neuropeptide receptors and beta-adrenergic receptors in human osteoblasts and human osteogenic sarcoma cells. Neurosci Lett 233: 125128, 1997.[CrossRef][ISI][Medline]
36. Valentijn K, Gutow AP, Troiano N, Gundberg C, Gilligan JP, and Vignery A. Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats. Bone 21: 269274, 1997.[CrossRef][ISI][Medline]
37. Vignery A and McCarthy TL. The neuropeptide calcitonin gene-related peptide stimulates insulin-like growth factor I production by primary fetal rat osteoblasts. Bone 18: 331335, 1996.[CrossRef][ISI][Medline]
38. Villa I, Dal Fiume C, Maestroni A, Rubinacci A, Ravasi F, and Guidobono F. Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity. Am J Physiol Endocrinol Metab 284: E627E633, 2003.
39. Villa I, Melzi R, Pagani F, Ravasi F, Rubinacci A, and Guidobono F. Effects of calcitonin gene-related peptide and amylin on human osteoblast-like cells proliferation. Eur J Pharmacol 409: 273278, 2000.[CrossRef][ISI][Medline]
40. Waugh DJ, Bockman CS, Smith DD, and Abel PW. Limitations in using peptide drugs to characterize calcitonin gene-related peptide receptors. J Pharmacol Exp Ther 289: 14191426, 1999.
41. Wimalawansa SJ. Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials. Endocr Rev 17: 533585, 1996.[ISI][Medline]
42. Wu D, Eberlein W, Rudolf K, Engel W, Hallermayer G, and Doods H. Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)2,7]hCGRP-preferring receptor. Eur J Pharmacol 40: 313319, 2000.
43. Yamaguchi DT, Hahn TJ, Beeker TG, Kleeman CR, and Muallem S. Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. J Biol Chem 263: 1074510753, 1988.
44. Yamaguchi DT, Hahn TJ, Iida-Klein A, Kleeman CR, and Muallem S. Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels. J Biol Chem 262: 77117718, 1987.