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
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ABSTRACT |
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cAMP; MAP kinase; preosteoblasts; Schild plot
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 2740% 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 CGRPs 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.
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MATERIALS AND METHODS |
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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 CGRP837 (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 CGRP837, [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 Students t-test. P < 0.05 values were considered significant.
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RESULTS |
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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.
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DISCUSSION |
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The more complete functional assays performed in our present study have provided evidence sufficient to modify the understanding of the interaction of CGRP837 and CGRP. CGRP837 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 CGRP837 and CGRPR subtypes: among four plots describing inhibition of CGRP-induced effects by CGRP837, 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 CGRP837, 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, CGRPs 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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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 CGRP837, 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 CGRPs 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 CGRPs 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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GRANTS |
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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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