Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)2,7] calcitonin gene-related peptide and CGRP8–37

Tomoyuki Kawase,1 Kazuhiro Okuda,2 and Douglas M. Burns3

1Division of Cellular Pharmacology, Department of Signal Transduction Research, and 2Division of Periodontology, Department of Oral Biological Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan; and 3Medical Research Service, Kansas City Department of Veterans Affairs Medical Center, and Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Missouri

Submitted 14 October 2004 ; accepted in final form 4 May 2005


    ABSTRACT
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 ABSTRACT
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Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)2,7]CGRP, and a relatively specific antagonist of CGRPR1, CGRP8–37. [Cys(Acm)2,7]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP8–37 potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.

cAMP; MAP kinase; preosteoblasts; Schild plot


THE NEUROPEPTIDE calcitonin gene-related peptide (CGRP) is widely distributed throughout the tissues of peripheral systems, including the cardiovascular and skeletal systems, where it produces multiple actions. It has been proposed that these diverse actions result from the activation of two CGRP receptor (CGRPR) subtypes: a subtype 1 CGRP receptor (CGRPR1) and a putative subtype 2 receptor (CGRPR2) (8, 24, 34). A molecular biological approach has revealed that CGRPR1 is a heterodimer composed of the calcitonin receptor-like receptor protein (CRLR or CLR) and receptor activity-modifying protein 1 (RAMP-1) (23). CGRPR1 is associated with the formation of cAMP through activation of adenylyl cyclase. In contrast, the putative CGRPR2 has been identified only in functional studies, in which receptor-mediated CGRP action is insensitive to the well-known antagonist of CGRPR1, CGRP8–37, but is instead mimicked by [Cys(Acm)2,7]CGRP (8, 34). Therefore, to our knowledge, the biochemical identity of a putative CGRPR2 has not yet been demonstrated using protein isolation, protein cross-linking studies, or molecular cloning.

A regulatory role for CGRP in skeletal tissue was originally suggested by the existence of prominent CGRP-positive peptidergic nerve fibers that permeate the marrow, lining, and mineralized areas of bone (3, 7, 12, 13, 33). Recent ultramorphological data have demonstrated close contact between these fibers and metaphyseal osteoblasts and osteoclasts in areas of active bone repair (14), and additional studies have suggested that CGRP-positive nerve fibers correlate with local centers of bone formation (4, 28). A recent study targeted transgenic expression of CGRP to osteoblasts and demonstrated a 27–40% increase in bone density at various skeletal sites and associated increases in cancellous bone formation and osteoblastic parameters (2). Most recently, a study with CGRP-specific knockout mice has demonstrated that generally decreased bone density and widespread osteopenia are due to decreased bone formation and concluded, "CGRP is a physiologic activator of bone formation" (27). In agreement with the latest findings, we have demonstrated in a series of in vitro studies examining dose-dependent activation of intracellular signaling pathways (6, 17, 18, 19, 22) that (immature) osteoblastic cells are one of the major targets for CGRP in skeletal tissue.

However, it is still unknown which CGRPR subtype mediates CGRP’s bone-forming activity. In our previous study (22), we demonstrated for the first time that a type of CGRPR1 is expressed in immature osteoblastic MG63 cells, but that this receptor did not display all of the properties expected for CGRPR1. Therefore, we could not exclude the possibility that another receptor subtype responsive to [Cys(Acm)2,7]CGRP also was expressed in these cells. To further examine this possibility, we have pharmacologically characterized receptor-mediated cellular responses in MG63 cells in more detail. Our results provide pharmacological evidence that at least two distinct subtypes of CGRPR with almost identical affinity to CGRP are expressed in MG63 cells.


    MATERIALS AND METHODS
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Cell and cell culture. Human osteosarcoma-derived immature osteoblastic MG63 cells were obtained from Dainippon Pharmaceutical (Osaka, Japan). MG63 cells were characterized in part by their pronounced cAMP response to CGRP and by providing a low cAMP response to parathyroid (PTH) hormone-related protein (22). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) in humidified 5% CO2-95% air at 37°C.

Measurement of intracellular cAMP. Cells were seeded onto 24-multiwell plates at a density of 4 x 104 cells/well and cultured for 2 days. Cells were preincubated overnight in 1% FBS-containing medium and then treated with CGRP (Peptide Institute, Osaka, Japan) or [Cys(Acm)2,7]CGRP (Bachem, Torrance, CA) for 15 min in the presence or absence of CGRP8–37 (Peptide Institute) or [Cys(Acm)2,7]CGRP in Hanks’ balanced salt solution (HBSS) containing 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma, St. Louis, MO) at 37°C. Intracellular cAMP was extracted and assessed using the cAMP enzyme immunoassay (EIA) system (Amersham Pharmacia Biotech, Little Chalfont, UK) as described previously (21).

Western blot analysis for MAPK phosphorylation. As described previously (20, 21), cells were treated with CGRP or [Cys(Acm)2,7]CGRP in combination with CGRP8–37, [Cys(Acm)2,7]CGRP, or IBMX. In some cases, cells pretreated with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 10 µM; Calbiochem) were used for CGRP stimulation. Cells were then lysed in Laemmli sample buffer and subjected to SDS-PAGE (9% linear gel) and immunoblot analysis using the following primary antibodies: polyclonal anti-phosphorylated-ERK antibody (1:1,000 dilution; New England BioLabs, Beverly, MA), polyclonal anti-ERK antibody (1:1,000 dilution; Transduction Laboratories or New England BioLabs), polyclonal anti-phosphorylated p38 MAPK antibody (1:1,000 dilution; New England BioLabs), or polyclonal anti-p38 MAPK antibody (1:3,000 dilution; Santa Cruz Biotechnology). Probed blots were then reacted with horseradish peroxidase (HRP)-conjugated protein A (1:10,000 dilution; Zymed, South San Francisco, CA), followed by visualization using the Amersham ECL system (Amersham Pharmacia Biotech). For reprobing with a different primary antibody, blots were stripped with Restore Western blot stripping buffer (Pierce, Rockford, IL).

The developed films were scanned using an Epson scanner (GTX-700; Epson, Tokyo, Japan) and quantitated using Scion Image (version 4.02; Frederick, MD) or Totallab (Nonlinear Dynamics, Newcastle upon Tyne, UK). The intensity of each phosphorylated protein band was normalized to the intensity of each total protein band observed in the lane.

Statistical analysis. Unless otherwise stated, results are expressed as means ± SD of at least three independent experiments. Using GraphPad Prism software (version 4 for Windows; GraphPad, San Diego, CA), we performed Schild plot analysis and calculated the values for half-maximal effective concentration (EC50) using nonlinear regression analysis. The 95% confidence interval was used for evaluation of statistically significant differences. In Fig. 5, for example, statistical analyses were performed using Student’s t-test. P < 0.05 values were considered significant.



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Fig. 5. Ability of IBMX or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) to inhibit or enhance CGRP-induced changes in p38 MAPK, CREB, or ERK phosphorylation. A: MG63 cells were treated with CGRP (0, 1, or 10 nM) for 15 min in the presence of IBMX (1 mM). B: cells were preincubated with H-89 (10 µM) for 30 min and then treated with CGRP (0, 1, or 10 nM) for 5 min. Experiments were repeated at least four times, and the results shown are representative of all of these data. C and D: each target protein was quantitated and plotted. Each column and vertical bar represents the mean ± SD of the data from more than three independent experiments. Significant differences were observed in CREB phosphorylation in the cases of both inhibitors and in ERK phosphorylation after preincubation with H-89 (aP < 0.05, bP < 0.02, cP < 0.002, dP < 0.001 vs. cultures treated with CGRP at same concentrations alone).

 

    RESULTS
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Effects of [Cys(Acm)2,7]CGRP and CGRP8–37 on cAMP production. We discovered in our previous study (22) that MG63 cells produce much greater increases in cAMP production in response to human CGRP (Fig. 1A) than are observed for many other osteoblastic cell lines (e.g., UMR106, SaOS-2). CGRP (10 pM–100 nM) dose dependently stimulated cAMP formation at 15 min in the presence of 1 mM IBMX, with a maximal 30- to 35-fold stimulation observed at 10 nM and with an EC50 of ~70 pM (Fig. 1A). The putative CGRPR2 agonist [Cys(Acm)2,7]CGRP used alone weakly stimulated cAMP production sixfold at 1 µM, while the CGRPR1 antagonist CGRP8–37 (up to 1 µM) had no effect when used alone. Coaddition of [Cys(Acm)2,7]CGRP (1 µM) effectively inhibited CGRP-induced cAMP production (Fig. 1A). As shown in Fig. 1B, comparative plots of CGRP8–37’s inhibition of CGRP at three selected concentrations (20 nM, 200 nM, and 2 µM) vs. CGRP alone demonstrated the expected shift to the right of the original dose-response curve. When this inhibitory effect was analyzed by performing more detailed Schild plot analysis, the pA2 (i.e., negative log of the antagonist concentration required to produce a twofold increase in the EC50 of the agonist) and the slope values for these linear regressions were calculated as 8.736 and 1.687, respectively.



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Fig. 1. A: dose-dependent effects of calcitonin gene-related peptide (CGRP) and peptide analogs on cAMP production in MG63 cells. Cells were treated for 15 min with the indicated concentrations of CGRP alone or in combination with 1 µM [Cys(Acm)2,7]CGRP, [Cys(Acm)2,7]CGRP alone, or CGRP8–37 alone in the presence of 3-isobutyl-1-methylxanthine (IBMX). cAMP production was evaluated as described in MATERIALS AND METHODS. B: dose-dependent inhibition of CGRP-induced cAMP production by CGRP8–37. Cells were treated for 15 min with CGRP alone in the presence of IBMX ({circ}) or with coaddition of CGRP8–37 at 20 nM ({triangleup}), 200 nM ({bullet}), or 2 µM ({blacksquare}). Each symbol and vertical bar represents the mean ± SD of data from four or more independent experiments. Inset: Schild plot analysis of data obtained from detailed functional inhibition studies using CGRP8–37. Resulting data were transformed into linear Schild plots using GraphPad Prism software.

 
Effects of [Cys(Acm)2,7]CGRP and CGRP8–37 on CREB phosphorylation. CGRP (0.1–100 nM) dose dependently phosphorylated cAMP response element-binding protein (CREB) (Fig. 2, AD), with an EC50 of ~260 pM (Fig. 2E), a value that is not as consistent as might have been expected with conclusions that CREB phosphorylation is regulated primarily by cAMP (25). [Cys(Acm)2,7]CGRP alone did not significantly stimulate CREB phosphorylation (Fig. 2, A and E). Coaddition of [Cys(Acm)2,7]CGRP (1 µM) substantially attenuated CGRP-induced CREB phosphorylation (Fig. 2B), but this antagonism was less complete than that observed for cAMP production (Fig. 2E vs. Fig. 1A). CGRP8–37 alone (up to 1 µM) did not show significant effects (Fig. 2, C and E), and as observed for cAMP formation, increasing concentrations of this antagonist effectively shifted the CGRP dose-response curve to the right (Fig. 2F). As shown, more detailed Schild plot analyses generated a pA2 value of 8.841 and a slope value of 1.258.



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Fig. 2. AD: dose-dependent effects of CGRP and peptide analogs, alone or in combination, on the phosphorylation of cAMP response element-binding protein (CREB) in MG63 cells. Cells were treated for 15 min with CGRP alone (AD), [Cys(Acm)2,7]CGRP alone (A), or CGRP8–37 alone (C). In addition, cells were treated with CGRP in the presence of 1 µM [Cys(Acm)2,7]CGRP (B) or 2 µM CGRP8–37 (D). Phosphorylated CREB (p-CREB) and total CREB were fractionated by performing SDS-PAGE and then visualized using Western blot analysis. Each experiment was repeated at least four times, and the results shown are representative of all of these data. E: dose-dependent effects of CGRP and CGRP analogs on CREB phosphorylation. Target bands were scanned, quantitated, and plotted as described in MATERIALS AND METHODS. F: dose-dependent inhibition of CGRP-induced CREB phosphorylation by CGRP8–37. Cells were treated for 15 min with CGRP in the absence ({circ}) or presence of CGRP8–37 at 20 nM ({triangleup}), 200 nM ({bullet}), or 2 µM ({blacksquare}). Each symbol and vertical bar represents the mean ± SD of data from more than four independent experiments. Inset: Schild plot analysis of data obtained from detailed functional inhibition studies using CGRP8–37.

 
Effects of [Cys(Acm)2,7]CGRP and CGRP8–37 on p38 MAPK phosphorylation. Findings very similar to those obtained for the phosphorylation of CREB were obtained for p38 MAPK phosphorylation. CGRP (0.1–100 nM) dose dependently phosphorylated p38 MAPK (Fig. 3, AD), with an EC50 of ~180 pM (Fig. 3, E or F). [Cys(Acm)2,7]CGRP alone failed to have an effect; however, coaddition of [Cys(Acm)2,7]CGRP potently but incompletely antagonized this action of CGRP (Fig. 3, A, B, and E). CGRP8–37 did not itself act as an agonist (up to 1 µM) and again effectively shifted the CGRP dose-response curve to the right at selected concentrations of 20 nM, 200 nM, and 2 µM (Fig. 3F). More detailed Schild plot analyses yielded a pA2 of 8.803 and a slope value of 1.212.



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Fig. 3. AD: dose-dependent effects of CGRP and peptide analogs, alone or in combination, on the phosphorylation of p38 MAPK in MG63 cells. Cells were treated for 15 min with CGRP alone (AD), [Cys(Acm)2,7]CGRP alone (A), or CGRP8–37 alone (C). Otherwise, cells were treated with CGRP in the presence of 1 µM [Cys(Acm)2,7]CGRP (B) or 2 µM CGRP8–37 (D). Experiments were repeated more than three times, and the results shown are representative of all of these data. E: dose-dependent effects of CGRP and CGRP analogs on CREB phosphorylation. F: dose-dependent inhibition by CGRP8–37 of CGRP-induced p38 MAPK phosphorylation. Cells were treated for 15 min with CGRP in the absence ({circ}) or presence of CGRP8–37 at 20 nM ({triangleup}), 200 nM ({bullet}), or 2 µM ({blacksquare}). Each symbol and vertical bar represents the mean ± SD of data from more than three independent experiments. Inset: Schild plot analysis of data obtained from detailed functional inhibition studies using CGRP8–37.

 
Effects of [Cys(Acm)2,7]CGRP and CGRP8–37 on ERK dephosphorylation. ERK dephosphorylation demonstrates a distinctly different pattern of peptide-receptor interaction. Both CGRP and [Cys(Acm)2,7]CGRP induced ERK dephosphorylation with similar maximal effects, although CGRP was approximately fourfold more potent than [Cys(Acm)2,7]CGRP (Fig. 4, A and E), with EC50 values of 210 and 820 pM, respectively. The potency and magnitude of CGRP-induced ERK dephosphorylation appeared almost identical to its effect on CREB or p38 MAPK phosphorylation. However, as predicted from earlier studies, in the presence of 1 µM [Cys(Acm)2,7]CGRP (the maximally effective concentration), CGRP could not produce a significant additional effect until concentrations of 100 nM were reached (Fig. 4E). Thus, because both peptides stimulate ERK dephosphorylation, it is difficult to evaluate the power of [Cys(Acm)2,7]CGRP to block CGRP action. Only at 1 µM and higher concentrations did CGRP8–37 alone slightly but significantly stimulate ERK dephosphorylation (Fig. 4E). Furthermore, the antagonistic actions of CGRP8–37 were substantially different in the case of ERK dephosphorylation than those observed for the three previously studied parameters: substantial antagonism did not occur until 2 µM CGRP8–37, and 1 µM CGRP still produced >50% of its maximal effect on ERK dephosphorylation in the presence of 2 µM CGRP8–37. Detailed Schild plot analyses of the partial inhibition produced by CGRP8–37 generated pA2 and slope values of 8.105 and 1.184, respectively.



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Fig. 4. AD: dose-dependent effects of CGRP and peptide analogs, alone or in combination, on ERK phosphorylation in MG63 cells. Cells were treated for 15 min with CGRP alone (AD), [Cys(Acm)2,7]CGRP alone (A), or CGRP8–37 alone (C). Otherwise, cells were treated with CGRP in the presence of 1 µM [Cys(Acm)2,7]CGRP (B) or 1 µM CGRP8–37 (D). Experiments were repeated more than three times, and the results shown are representative of all of these data. E: dose-dependent effect of CGRP and CGRP analogs on ERK phosphorylation. F: dose-dependent inhibition of CGRP-induced ERK dephosphorylation by CGRP8–37. Cells were treated for 15 min with CGRP alone ({circ}) or with coadditions of CGRP8–37 at 20 nM ({triangleup}), 200 nM ({bullet}), or 2 µM ({blacksquare}). Each symbol and vertical bar represents the mean ± SD of data from at least four independent experiments. Inset: Schild plot analysis of data obtained from detailed functional inhibition studies using CGRP8–37.

 
Comparison of computer-calculated 95% confidence intervals for each data plot demonstrated the pA2 value of ERK dephosphorylation to be statistically distinguishable from that obtained for the other three plots. Upon initial inspection, the slope of the ERK Schild plot appeared substantially different from that of all of the other plots; however, analysis using the 95% confidence interval comparison revealed that this value was significantly different from only the slope of the cAMP Schild plot.

Effects of IBMX and H-89 on CGRP action. To examine further whether different receptor subtypes are involved in each response, pharmacological inhibitors were applied to this experimental system. As shown in Fig. 5, A and C, coaddition of 1 mM IBMX, a level sufficient to inhibit phosphodiesterase-dependent degradation of cAMP in these cells (Kawase T, Okuda K, and Burns DM, unpublished observation), to 15-min CGRP treatments clearly augmented CGRP-induced CREB phosphorylation and slightly enhanced CGRP-induced ERK dephosphorylation. However, IBMX had no appreciable effect on CGRP-induced p38 MAPK phosphorylation. In support of this finding, when cells were pre- or coincubated with 10 µM H-89, a relatively specific inhibitor of cAMP-dependent protein kinase, CGRP-induced CREB phosphorylation was substantially blocked after 5 min but p38 MAPK phosphorylation was unaffected (Fig. 5, B and D). Despite having observed little effect for added IBMX, coaddition of H-89 significantly attenuated CGRP-induced ERK dephosphorylation.


    DISCUSSION
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How many functional CGRPR subtypes are expressed in MG63 cells? Until now, it has been widely accepted that CGRPR1 is the predominant CGRPR subtype expressed in osteoblastic cells. In support of this concept, our previous study (22) demonstrated in radioligand receptor binding assays, followed by both directly binding kinetic and Scatchard analyses, that only one type of CGRP-binding site is expressed on MG63 cells, and in immunocytochemical assays that both the main protein components of a CGRPR1 (i.e., CLR and RAMP-1) are present in MG63 cells. Therefore, it seemed likely that MG63 cells predominantly express a CGRPR1 (or at least a variation of this receptor subtype). Nonetheless, we also observed that a 200-fold excess of CGRP8–37, a specific antagonist of CGRPR1, competes for only 70–80% of membrane-bound fluorescein-conjugated CGRP (22) and that 1 µM CGRP8–37 antagonizes only lower concentrations (~1 nM) of CGRP in cellular dephosphorylation of ERK (22). We speculated that incomplete competition could be attributed to the large difference in binding affinities between these individual peptides, but we could not completely rule out the possibility that a second receptor with a very similar CGRP-binding affinity might be expressed in MG63 cells.

The more complete functional assays performed in our present study have provided evidence sufficient to modify the understanding of the interaction of CGRP8–37 and CGRP. CGRP8–37 potently antagonized CGRP-induced phosphorylation of CREB and p38 MAPK as observed in cAMP production, but could not produce complete inhibition of ERK dephosphorylation induced by higher concentrations (~1 µM) of CGRP. Schild plot analyses clearly showed two distinct sets of interactions between CGRP8–37 and CGRPR subtypes: among four plots describing inhibition of CGRP-induced effects by CGRP8–37, the pA2 value of the Schild plot regression for the partial inhibition of ERK dephosphorylation appeared significantly different. The Schild plot for cAMP formation is statistically different from that for ERK dephosphorylation in both pA2 and slope values.

Complementary findings were obtained in assays using [Cys(Acm)2,7]CGRP. As described in the introduction, [Cys(Acm)2,7]CGRP has been widely accepted as an agonist of putative CGRPR2 (8, 34) and its antagonism of what appeared to be CGRPR1-mediated effects was not expected (15). In the present study, [Cys(Acm)2,7]CGRP did not act exactly as data in the literature would have predicted. [Cys(Acm)2,7]CGRP produced substantial agonistic effects only on dephosphorylation of ERK and instead potently antagonized CGRP-induced cAMP production and the phosphorylation of CREB and p38 MAPK. Taken together with the data obtained from CGRP8–37, therefore, we must revise our previous conclusions and now propose a novel dual receptor explanation that immature osteoblastic MG63 cells possess two similar but distinguishable subtypes of CGRPR. Presumably, CGRP’s effects on cAMP, CREB, and p38 MAPK are associated primarily with CGRPR1 (or a CGRPR1 variant), while CGRP-induced ERK dephosphorylation is associated with a CGRPRx (see Fig. 6).



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Fig. 6. Proposed mechanism of interaction between CGRP and its specific receptor subtypes to produce the four major cellular responses addressed in this paper. Our best explanation for the data in this report is that CGRP acts on two distinct CGRP receptor (CGRPR) subtypes expressed by MG63 cells, but these receptor subtypes share some intracellular signaling pathways. 1) cAMP is produced mainly through activation of a CGRPR1 variant, but the agonistic action of [Cys(Acm)2,7]CGRP indicates the additional involvement of a second CGRPR subtype (CGRPRx) in this response. 2) Because CGRP-induced p38 MAPK phosphorylation was potently blocked by CGRP8–37, as was cAMP production, this response is produced predominantly through activation of CGRPR1. However, because cAMP-related inhibitors (i.e., IBMX, H-89) had no appreciable effect, this response is not mediated by cAMP. 3) Because CREB phosphorylation, which is known to depend predominantly on cAMP (25), was potently inhibited by CGRP8–37 and weakly stimulated by [Cys(Acm)2,7]CGRP, this response was mediated by a cAMP-dependent mechanism mainly through activation of CGRPR1 and secondarily by activation of CGRPRx. 4) Because CGRP-induced ERK dephosphorylation could not be inhibited effectively by high levels of CGRP8–37, which potently inhibited CGRP-induced cAMP production and p38 MAPK or CREB phosphorylation in parallel experiments, this response is mediated primarily through activation of CGRPRx. However, this response was also slightly and significantly influenced by IBMX and H-89, respectively, and thus we cannot exclude the possible additional involvement of CGRPR1. (Note that the thicker of any two arrows represents the major pathway.)

 
Skepticism about the traditional classification of CGRPR subtypes. It should be noted that our treatment of these topics is based primarily on classically published data suggesting the widely accepted general concept that two or more versions of a CGRP receptor do exist. However, a series of recent and extensive studies by Abel and coworkers (26, 32) raised a cautionary note arguing against those criteria established for the classification of CGRPR subtypes (8, 24, 34). In CGRPR1-transfected human embryonic kidney (HEK)-293 cells (26), [Cys(Acm)2,7]CGRP binds to this receptor and displaces [125I]CGRP with a Ki of 28–32 nM as did another putative CGRPR2 agonist, [Pro14]CGRP (Ki of 5.5–9 nM), and this agonist is capable of stimulating cAMP production. For these puzzling effects, therefore, Abel and colleagues offered possible explanations that the effectiveness of various CGRPR2 agonists in different tissues (8, 24, 34) is actually a function of the quantity of excess CGRPR1 being held in cellular reserve (26, 32), and an explanation for tissue-to-tissue differences in the effects of agonists and antagonists is that this behavior reflects the local complement of proteases present to degrade the unprotected peptides (26). In addition, the affinities of agonists and antagonists are possibly affected by whether the peripheral membrane receptor component protein (10) is present in the receptor complex (26).

Our data could prove to be consistent with these interpretations, because our previous (22) and present studies have demonstrated pharmacologically distinguishable CGRPR subtypes in MG63 cells that show a very similar affinity to [125I]CGRP. Nonetheless, we have not yet obtained direct evidence to strongly support or demonstrate this explanation. Therefore, a considerable body of additional experiments is needed to provide evidence that distinguishes one of several candidates thought to be expressed in MG63 cells that may serve as a functional CGRPR2 (or "CGRPRx" as shown in Fig. 6).

How are receptor subtypes associated by signaling pathways with cellular responses in MG63 cells? It seems possible to classify these cellular responses by the degree to which the cAMP-dependent pathways are involved. IBMX augmented CGRP-induced CREB phosphorylation but not p38 MAPK phosphorylation. Furthermore, H-89 blocked CGRP-induced CREB phosphorylation and ERK dephosphorylation but not p38 MAPK phosphorylation. Therefore, these findings suggest that both CREB and p38 MAPK phosphorylation are apparently mediated by the same receptor subtype (i.e., a CGRPR1 variant that stimulates cAMP production), but only CREB phosphorylation is located downstream of the cAMP-mediated pathway. The other receptor subtype (shown as CGRPRx in Fig. 6) on which [Cys(Acm)2,7]CGRP acts as an agonist weakly activates adenylyl cyclase to produce cAMP and strongly stimulates ERK dephosphorylation through a mechanism that is primarily cAMP independent.

Furthermore, we recently reported that CGRP induces biphasic increases in intracellular free Ca2+ concentration ([Ca2+]i): an initial transient increase and a subsequent slow incremental increase in MG63 cells (6). The slow increment in [Ca2+]i is completely blocked by a cAMP antagonist, Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate Rp-cAMPS, or verapamil, while the transient initial peak is sensitive only to thapsigargin. These data primarily suggest that the CGRPR is associated with two independent components, such as a cAMP-operated Ca2+ channel and a component responsible for Ca2+ discharge from intracellular stores such as inositol 1,4,5-trisphosphate. In consideration of the finding that the slow increment is also sensitive to CGRP8–37, these data also support the concept that in addition to the known CGRPR1, MG63 cells possess a distinct second receptor subtype (i.e., CGRPRx as shown in Fig. 6).

Reevaluation of the biological significance of CGRP on osteoblastic bone formation. With regard to CGRP’s action on osteoblastic cells, we have demonstrated in several publications (5, 22) that CGRP stimulates biomineralization and expression of type I collagen mRNA in cultures of osteogenic cells. As described in the introduction of this report, recent molecular biological approaches using transgenic animals have demonstrated in vivo anabolic actions of CGRP (2, 27). Therefore, there can no longer be real doubt that CGRP plays an important role in bone formation.

The biological significance of the intracellular signaling pathways invoked by CGRP in osteogenic cells is suggested by several recent studies. Bone morphogenetic protein-2 activates p38 MAPK to induced osteoblastic differentiation (11). Prostaglandin E1 enhances alkaline phosphatase activity through activation of p38 MAPK and JNK by a cAMP-dependent mechanism in osteoblastic MC3T3-E1 cells while ERK is dephosphorylated (16). Oxidative stress inhibits osteoblastic differentiation of calvarial osteoblasts through activation of ERK (1), and glucocorticoids, useful cofactors to stimulate the osteogenic differentiation of marrow mesenchymal stem cells, rapidly stimulate ERK dephosphorylation and consequently impair the proliferation of osteoblastic cells (9). In addition, cAMP production, which has classically been used as a marker response of osteoblastic cells to PTH, recently has been shown to be required for c-fos expression, which is thought to have many pleiotropic and essential effects in bone, through phosphorylation of CREB (30). Therefore, we think it very likely that this complex of the individual signaling pathways may prove central to understanding CGRP action in osteogenic bone cells and bone formation.

To our knowledge, it is still widely accepted, without much in the way of direct convincing evidence, that CGRPR1 is the predominant functional receptor for CGRP in osteoblastic cells throughout their differentiated pathways. Several research groups have suggested the possibility that osteoblastic CGRPR1 gradually disappears with the maturity of cell differentiation (29, 31). On the basis of our indirect data from years of functional assays in a variety of cell lines, we also advanced a similar possibility (17). Although the situation may ultimately prove more complex, we would now simply suggest that the second CGRP subtype is important to CGRP’s bone anabolic effects. We hope that our present data provide some guidance for reevaluating the biological significance of CGRP in osteoblastic bone formation.

Our present pharmacological data support the possibility that a second subtype of CGRPR is expressed in preosteoblastic MG63 cells. However, previously published results from our [125I]CGRP binding assay indicate that this additional receptor subtype is not identical to the classic CGRPR2 described in other tissues and cell types and may instead represent a variant of CGRPR2.


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 ABSTRACT
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This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, by a Merit Review Grant and Research Career Scientist Award from the U.S. Department of Veterans Affairs, and by a Lied Bridge Funds Grant from the University of Kansas Medical Center Research Institute.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kawase, Division of Cellular Pharmacology, Dept. of Signal Transduction Research, Graduate School of Medical and Dental Sciences, Niigata Univ., 2-5274 Gakkocho-dori, Niigata 951-8514, Japan (e-mail: kawase{at}dent.niigata-u.ac.jp)

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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 ABSTRACT
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