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The extracellular calcium (Ca2+o)-sensing receptor is expressed in myeloma cells and modulates cell proliferation
BBRC Biochemical and Biophysical Research Communications 299 (2002) 532–538 www.academicpress.com
The extracellular calcium (Ca2o+)-sensing receptor is expressed in myeloma cells and modulates cell proliferation Toru Yamaguchi,a,* Mika Yamauchi,b Toshitsugu Sugimoto,b Dharminder Chauhan,c Kenneth C. Anderson,c Edward M. Brown,d and Kazuo Chiharab a Department of Internal Medicine, Takatsuki General Hospital, 1-3-13 Kosobe-cho, Takatsuki 569-1192, Japan Division of Endocrinology/Metabolism, Neurology, and Hematology/Oncology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan c Division of Hematologic Malignancies, Department of Medicine, Dana-Farber Cancer Institute, Boston, MA, USA Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA b
d
Received 25 October 2002
Abstract The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2þ o ) homeostasis by enabling parathyroid, kidney, and other cells to directly ‘‘sense’’ changes in Ca2þ . In multiple myeloma-associated o bone disease, myeloma cells could raise the level of Ca2þ o within their immediate vicinity in the bone marrow microenvironment, through their known capacity to cause bone destruction by stimulating osteoclastic bone resorption. Thus if myeloma cells expressed the CaR, they might sense these locally elevated levels of Ca2þ o , which could, in turn, potentially modify their function(s) in ways that could contribute to myeloma bone disease or other aspects of the pathophysiology of this disabling hematological malignancy. In this study, we examined the expression of the CaR in three myeloma cell lines, human U266, IM-9, and RPMI8226 cells. CaR protein was present in all three cell lines as assessed by immunocytochemistry and Western blot analysis using a monoclonal antibody specific for the CaR. Moreover, the use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in the three cell lines. Exposure 3þ to known polycationic agonists of the CaR, including high Ca2þ o (2.5 mM), neomycin, and gadolinium (Gd ) as well as a specific CaR activator, NPS R467, augmented cell proliferation in all three cell lines. RT-PCR revealed that U266 cells, but not IM-9 cells or RPMI8226 cells, expressed interleukin-6 (IL-6), the expression of which was not enhanced by treatments with CaR agonists. Therefore, taken together, our data first document the fact that the myeloma cell lines, U266, IM-9, and RPMI8226, all express CaR protein and mRNA. Moreover, the CaR expressed on myeloma cells could sense the locally high levels of Ca2þ o in the vicinity of sites of osteoclastic bone resorption and stimulate their proliferation in an IL-6-independent manner. These processes may result in promoting further growth of the tumor and aggravating the associated bone disease. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Calcium-sensing receptor; Multiple myeloma; U266 cells; IM-9 cells; RPMI8226 cells; Proliferation
Myeloma bone disease is characterized by bone destruction that can produce intractable bone pain; it is caused by bone destruction stimulated by tumor-derived factors that activate osteoclastic bone resorption [1,2]. In most patients, there are multiple discrete lytic lesions adjacent to the site of deposits or nests of myeloma cells, caused by osteoclasts that accumulate and resorb bone in response to tumor-derived products. In patients with * Corresponding author. Fax: +81-726-82-3834. E-mail address: [email protected] (T. Yamaguchi).
myeloma, macrophage inflammatory protein-1a (MIP1a) produced by myeloma cells, in combination with receptor activator of nuclear factor jB ligand (RANKL) and interleukin-6 (IL-6) that are produced by marrow stromal cells in response to myeloma cells, enhances osteoclast formation through their combined effects on osteoclast precursors [3,4]. Hypercalcemia occurs as a consequence of increased osteoclastic bone resorption in about one-third of patients with advanced disease. The levels of the extracellular calcium concentration (Ca2þ o ) underneath resorbing osteoclasts are known to
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 6 9 0 - 6
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reach levels as high as 8–40 mM [5]. Thus it is likely that myeloma cells close to activated osteoclasts are exposed to locally high levels of Ca2þ o in the microenvironment surrounding destructive bone lesions. If these cells possessed the capacity to sense these changes in Ca2þ o , their functional properties might be modulated in ways that could contribute to the pathophysiology of multiple myeloma and its associated bone disease. One candidate for a Ca2þ o -sensing mechanism that could be expressed on myeloma cells is the Ca2þ o -sensing receptor (CaR), which has been cloned from bovine and human parathyroid gland [6,7], rat kidney [8], and thyroid C-cells [9]. The central, non-redundant role of the CaR in humans has been documented by showing that inactivating and activating mutations of the CaR gene cause inherited hyper- and hypocalcemic disorders [10,11], rendering affected family members inappropriately ÔresistantÕ or Ôsensitive,Õ respectively, to the usual effects of Ca2þ o on parathyroid and renal functions. The 3þ CaR binds its cationic ligands, such as Ca2þ o , Gdo , and neomycin, with EC50 values of 3 mM, 20 lM, and 60 lM, respectively. These agonists produce a G protein-dependent activation of intracellular second messenger pathways, including phospholipase C (PLC), leading to elevations in the levels of inositol trisphosphate (IP3 ) and the cytosolic calcium concentration [6]. In a previous study, we found that a variety of cell types within bone marrow express the CaR [12] that are not directly involved in forming or breaking down bone (e.g., the functions of osteoblasts and osteoclasts, respectively). In the marrow, hematopoietic precursors expressing the CaR would likely experience significant variations in the levels of Ca2þ o to which they are exposed as a result of changes in the prevailing rate of bone turnover within the local bone/bone marrow microenvironment. These cells include red blood cell precursors, megakaryocytes (the precursors of blood platelets), monocytes, and macrophages, all of which express relatively high levels of the CaR, and white blood cells precursors, which express lower levels of the receptor [12]. Since myeloma cells also arise from hematopoietic precursors (e.g., plasma cells), it is possible that the CaR is expressed in myeloma cells and can sense locally high levels of Ca2þ o within osteolytic bone lesions in myeloma patients. In this study, therefore, we used the three human myeloma cell lines, U266, IM-9, and RPMI8226 cells, to examine the expression of the CaR in myeloma cells and to assess its role in regulating their proliferation. Materials and methods Materials. All routine culture media were obtained from Invitrogen Japan K.K. (Tokyo, Japan). All chemicals were of the highest grade available commercially. The CaR activator, NPS R-467 [13], was provided by NPS Pharmaceuticals (Salt Lake City, UT).
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Cell culture. The U266 human multiple myeloma-derived cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). The IM-9 and RPMI8226 human multiple myelomaderived cell lines were obtained from the Japanese Collection of Research Bioresources (Sen-nan, Japan). Each cell line was grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin in 5% CO2 at 37 °C. The medium was changed twice weekly and the cells were subcultured in 75 cm2 culture flasks. For morphological evaluation, cells were removed from the flasks, pelleted by centrifugation, and resuspended in phosphate-buffered saline (PBS). The cell suspension was spread on a glass microscope slide, air-dried, fixed with 4% formaldehyde in PBS for 5 min, and washed with PBS. The slides were stored at 4 °C until assessment for the presence of the CaR as described below. Immunocytochemistry for CaR in U266, IM-9, and RPMI8226 cells. A mouse anti-CaR monoclonal antibody ADD was provided by NPS Pharmaceuticals, Salt Lake City, UT. This antibody was raised against a peptide (ADD) corresponding to amino acids 214–235 of the human CaR, which resides within the predicted aminoterminal extracellular domain of the receptor. The antibody was used for immunocytochemistry and Western blot analysis as described below. Fixed U266, IM-9, and RPMI8226 cells were treated with DAKO Protein Block Serum-Free Solution (DAKO Corp.) for 1 h and then incubated overnight at 4 °C with the anti-CaR antibody (ADD) at a concentration of 5 lg/ml in blocking solution. Negative controls were carried out by incubating the cells with the anti-CaR antibody (5 lg/ml) that had been preabsorbed with 10 lg/ml of the ADD peptide. After washing the cells three times with 0.5% bovine serum albumin in PBS for 10 min each, alkaline phosphatase-coupled, goat anti-mouse IgG (1:200; Sigma Chemical, St. Louis, MO) was added and incubated for 1 h at room temperature. The cells were then washed with PBS three times for 10 min each and the color reaction was developed for 10–20 min using a solution consisting of 44 ll nitroblue tetrazolium chloride (NBT) (75 mg/ml) and 33 ll 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) (50 mg/ml) in 10 ml of 0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2 , and 1 mg/ml levamisole, which was included to inhibit endogenous cellular alkaline phosphatase (ALP) activity. The color reaction was stopped by washing the slides twice in the above solution without NBT or BCIP and then twice in water. Western blot analysis of CaR in U266, IM-9, and RPMI8226 cells. U266, IM-9, and RPMI8226 cells in 75 cm2 culture flasks were removed from the flasks, pelleted by centrifugation, resuspended in 1 ml of a lysis solution (1% SDS, 10 mM Tris–HCl, pH 7.4), and heated to 65 °C. The cells were homogenized by brief sonication and heated for an additional 5 min at 65 °C. Insoluble material was removed by centrifugation for 5 min. The resultant whole cell lysate in the supernatant was stored at )20 °C until Western blot analysis was carried out. Aliquots of 75 lg of protein were dissolved in SDS–Laemmli gel loading buffer containing 100 mM dithiothreitol, incubated at 37 °C for 15 min, and resolved electrophoretically on 6.5% SDS–polyacrylamide gels. Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon Transfer; Nihon Millipore, Tokyo, Japan) at 240 mA for 40 min in transfer buffer containing 19 mM Tris– HCl, 150 mM glycine, 0.015% SDS, and 20% methanol. The blots were then blocked with 1% BSA in PBS containing 0.25% Triton X-100 (blocking solution) for 2 h and incubated with ADD or with peptideblocked antibody [the same amount of antibody preincubated at room temperature for 60 min with twice the amount (wt/wt) of the synthetic CaR peptide against which it was raised] at a concentration of 1 lg/ml in the blocking solution overnight at 4 °C. The blots were then washed three times with PBS containing 0.25% Triton X-100 (washing solution) at room temperature for 10 min each. The blots were further incubated with a 1:2000 dilution of ALP-coupled, goat anti-mouse IgG (Sigma Chemical Co.) in the blocking solution for 1 h at room temperature. The blots were then washed three times with washing solution at room temperature for 10 min each and specific protein bands were detected using NBT and BCIP as described above.
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PCR amplification of CaR transcript(s) in U266, IM-9, and RPMI8226 cells. Total RNA was prepared from U266, IM-9, and RPMI8226 cells with the TRIzol Reagent (Invitrogen). Total RNA (1 lg) was used for the synthesis of single-stranded cDNA (cDNA synthesis kit, Invitrogen). The resultant first-stranded cDNA was used for the PCR procedure. PCR was performed at a final concentration of 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl2, 0.2 mM dNTP, 0.4 lM of forward primer, 0.4 lM of reverse primer, and 1 ll of ELONGASE enzyme mix (a Taq/Pyrococcus species GB-D DNA polymerase mixture) (Invitrogen). The primer sequences used were sense primer, 50 -AAGCACCTACGGCATCTAA-30 (nucleotides 1384–1402 of the human CaR) and antisense primer, 50 -GCGATCCC AAAGGGCTCCG-30 (nucleotides 1826–1844), according to the method of Goebel et al. [14]. These were intron-spanning primers to avoid confusion arising from amplification of the same sequences within genomic DNA. The PCR for the human CaR was run under the following conditions: initial denaturation at 94 °C for 5 min, 38 cycles of amplification (50-s denaturation at 94 °C, 50-s annealing at 50 °C, and 2-min extension at 72 °C). The reaction was completed with an additional 8-min incubation at 72 °C to allow completion of extension. PCR products were fractionated on 1.2% agarose gels. The presence of a 461 nucleotide-long amplified product was indicative of a positive PCR arising from cDNA. The PCR product in the reaction mixture was purified using the QIAquick PCR purification kit (Qiagen, Santa Clarita, CA) and subjected to direct, bi-directional sequencing employing the same primer pairs used for PCR by means of an automated sequencer (AB377, Applied Biosystems, Foster City, CA) using dideoxy terminator Taq technology. Assays of cell proliferation. Proliferation of myeloma cells was assessed by counting cell numbers using a hemocytometer or by a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay is a colorimetric assay used to determine cell number and/or assess cellular viability, based on the ability of mitochondria in live cells to oxidize thiazolyl blue, a tetrazolium salt. One hundred ll each of the U266, IM-9, and RPMI8226 cell lines in the growth media was seeded in 96-well plates at a density of 1 104 cells/ml. After incubating for appropriate periods and removing medium for counting cell numbers, 10 ll MTT (10.4 mg/ml) was added to each well containing 40 ll of myeloma cells. The plates were incubated at 37 °C in humidified 5% CO2 for 4 h to convert water-soluble MTT to insoluble formazan, which occurs only in viable cells. After 4 h, 100 ll of 50% dimethylformamide with 20% SDS was added to each well to solubilize formazan and the plates were incubated at 37 °C overnight. Absorbance was then measured at 595 nm with background subtraction at 655 nm. PCR amplification of IL-6 in U266, IM-9, and RPMI8226 cells. U266, IM-9, and RPMI8226 cells were incubated with serum-free PRPMI1640 medium for 24 h in the absence or presence of Ca2þ 2.5 mM or NPS R467 1 lM. After the preparation of total RNA and the synthesis of singlestranded cDNA, the PCR was performed as described above. The primer sequences used for human IL-6 were sense primer, 50 -CATCCTCGACG GCATCTCAG-30 and antisense primer, 50 -GTCCTAACGCTCATA CTTTT-30 . The PCR was run by 27 cycles of amplification (30-s denaturation at 94 °C, 30-s annealing at 52 °C, and 1-min extension at 72 °C) and an additional 10-min incubation at 72 °C. PCR products were fractionated on 1.2% agarose gels. The presence of a 582 nucleotide-long amplified product was indicative of a positive PCR arising from cDNA. The PCR for GAPDH was also performed as a control.
Results Immunoreactivity of CaR protein in U266, IM-9, and RPMI8226 cells using CaR-specific antibody To clarify whether the CaR is expressed in myeloma cells, we investigated the presence of the receptor in
Fig. 1. Immunocytochemistry of U266, IM-9, and RPMI8226 cells carried out as described in Materials and methods using a monoclonal CaR-specific antibody (ADD). Immunocytochemistry revealed moderate to strong CaR staining in the three cells (upper panel), which were eliminated by preincubating the primary antibody with the peptide against which it was raised (lower panel). The photomicrographs were taken at a magnification of 400.
three myeloma cell lines, U266, RPMI8226, and IM-9. Immunocytochemistry of these cells with a CaR-specific monoclonal antibody revealed moderate to strong CaR staining (Fig. 1, upper panel), which was eliminated by preincubating the primary antibody with the peptide against which it was raised (Fig. 1, lower panel). We also performed Western blot analysis on proteins isolated from U266, RPMI8226, and IM-9 cells (Fig. 2, left). In proteins in total cellular lysates obtained from the three cell lines, the ADD stained double bands at
Fig. 2. Left: Western blot analysis of whole cell lysates from U266, IM9, and RPMI8226 cells performed as described in Materials and methods. Double bands at molecular weights of 160 kDa that were stained in the presence of specific antibody were consistent with the intact, glycosylated CaR. The specificity of the labeling by the antiCaR antibody used in this study was confirmed by reduction or abolition of the bands in extracts of these cells when they were incubated with antibody preabsorbed with the peptide against which it was raised. Right: Analysis of CaR transcripts in U266, IM-9, and RPMI8226 cells by RT-PCR, performed as described in Materials and methods. The product was of the expected size, 461 bp, for a product derived from authentic CaR transcript(s). No products were observed when the reverse transcriptase (RT) was omitted during synthesis of cDNA.
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molecular weights of 160 kDa-sizes consistent with the intact glycosylated human CaR [15,16]. These bands were specific, since it was markedly diminished in intensity following preabsorption of the primary antibody with its specific peptide. Detection of CaR mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR RT-PCR with CaR-specific, intron-spanning primers, which were utilized to ensure that the CaR products was not the result of amplification of contaminating genomic DNA segments, amplified products of the expected size from U266, IM-9, and RPMI8226 cells, 461 bp (Fig. 2, right). No products were observed when the reverse transcriptase (RT) was omitted during synthesis of cDNA. DNA sequence analysis of the PCR products revealed 100% identities with the corresponding region of the human parathyroid CaR cDNA sequences [7] (not shown). These results show that the RT-PCR
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product corresponded to an authentic CaR sequence, indicating the presence of bona fide CaR transcript(s) in these cells. The effects of CaR agonists on the proliferation of U266, IM-9, and RPMI8226 cells When cell numbers were counted, high Ca2þ o (2.5 mM), neomycin sulfate (100 lM) and Gd3þ o (25 lM), polycationic agonists of the CaR, as well as NPS R467 (1 lM), a specific CaR activator, significantly stimulated the proliferation of U266, IM-9, and RPMI8226 cells, up to treatments for 3 days (Fig. 3) (p < 0:05). The MTT assay performed after treatments for 1 day also showed that the CaR agonists significantly stimulated the proliferation of the three cells (p < 0:05) (Fig. 4). The effects of CaR agonists on the expression of IL-6 mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR We performed RT-PCR for IL-6 in U266, IM-9, and RPMI8226 cells in order to examine whether or not CaR agonists exert their mitogenic effect on the cells by enhancing the expression of endogenous IL-6 in the cell
Fig. 3. Effects of CaR-agonists on the proliferation of U266, IM-9, and RPMI8226 cells. Cell numbers were counted up to 3 days under treatments with various CaR-agonists as described in Materials and methods. Each bar represents the mean SEM for nine determinations. * p < 0:05 compared to cells treated with 0.5 mM Ca2þ o .
Fig. 4. Effects of CaR-agonists on the proliferation of U266, IM-9, and RPMI8226 cells. The MTT assay was performed after 1 day treatments of the cells with CaR-agonists as described in Materials and methods. Each bar represents the mean SEM for eight determinations. * p < 0:05 compared to cells treated with 0.5 mM Ca2þ o .
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Fig. 5. The effects of CaR agonists on the expression of IL-6 mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR, performed as described in Materials and methods. U266 cells expressed IL-6 under a basal Ca2þ condition (0.5 mM), but the expression was not enhanced by o treatments with high Ca2þ o (2.5 mM) or NPS R467 (1 lM). IL-6 was not expressed in IM-9 cells or RPMI8226 cells under either basal Ca2þ o or CaR agonist-treated conditions.
lines (Fig. 5). Although U266 cells expressed IL-6 under a basal Ca2þ o condition (0.5 mM), the expression was not augmented by the treatment with high Ca2þ o (2.5 mM) or NPS R467 (1 lM). IL-6 was not expressed in IM-9 cells or RPMI8226 cells under either basal Ca2þ o or CaR agonist-treated conditions.
Discussion Our results showed that U266, IM-9, and RPMI8226 cell lines clearly expressed CaR protein by immunocytochemistry (Fig. 1) as well as by Western blot analysis (Fig. 2, left), which revealed specific double bands at molecular weights consistent with those of the intact, glycosylated CaR [15,16]. In addition, RT-PCR performed on total RNA isolated from these three cell lines followed by sequence analysis of the PCR products indicated the presence of bona fide CaR transcripts (Fig. 2, right). Thus, the present study shows that these myeloma cell lines express both CaR protein and mRNA and the receptor could potentially sense local changes in Ca2þ related to excessive osteoclastic bone resorption o associated with myeloma bone disease. The functions of the CaR in hematopoietic cells, including myeloma cells, are not well understood. There is
a limited body of data, however, indicating that changes have direct actions on these CaR-expressing in Ca2þ o hematopoietic elements that are of potential physiological relevance. Elevated levels of Ca2þ o have several effects on macrophages or monocytes. First, high Ca2þ o promotes chemotaxis of peripheral blood monocytes [17]; therefore, Ca2þ and the CaR share some of the o properties of chemokines and chemokine receptors, respectively [18]. Second, Bornefalk et al. [19] have shown that high Ca2þ o stimulates the secretion of IL-6 both in vivo and in vitro from peripheral blood monocytes. Finally, raising Ca2þ o potentiates the fusion of rat alveolar macrophages induced by 1,25-dihydroxyvitamin D3 [20]. Elevating Ca2þ also stimulates the formation of o erythroid colonies in vitro and raises the cytosolic calcium concentration in erythroid precursors isolated from uremic patients, an effect that is potentiated by 1,25ðOHÞ2 D3 [21], which is known to upregulate CaR expression [22]. Ca2þ o also modulates several processes in platelets, including stimulating the release of arachidonic acid [23] and inhibiting the accumulation of cAMP [24]—effects that might potentially be CaR-mediated. Interestingly, Ca2þ o has been quantified directly in platelet clumps during platelet activation and it decreases to a substantial extent [25]. Thus while much additional work needs to be carried out defining CaRÕs potential roles in cells of various hematopoietic lineages, available data suggest that (a) several of these cells express the CaR, (b) Ca2þ o modulates their functions, and (c) Ca2þ can change within their local microenvirono ments—either within the bone marrow as a function of bone turnover or within peripheral blood (e.g., within clumps of activated platelets). IL-6 is proposed to be either an autocrine or paracrine growth factor for some human myeloma cells [26– 28]. Kawano et al. [28] have postulated an autocrine growth mechanism because myeloma cells secrete IL-6 and proliferate in response to this cytokine in vitro. More recently, myeloma cells have been triggered via their cell surface CD40 to both secrete IL-6 and to proliferate [29,30], suggesting the potential for induction of IL-6-mediated autocrine growth in myeloma. The possible role of IL-6-mediated paracrine control of myeloma cell growth is supported by observations that (a) bone marrow stromal cells are the major source of IL-6 in myeloma [27,31,32], (b) freshly isolated myeloma cells cultured without exogenous IL-6 rapidly stop proliferating [33], and (c) adhesion of myeloma cells to stromal cells up-regulates IL-6 secretion by stromal cells [34,35]. In this study, we found that CaR agonists stimulated proliferation of U266, IM-9, and RPMI8226 cells (Figs. 3 and 4). However, the mechanisms by which CaR agonists exert a mitogenic effect on myeloma cells seemed to be independent to IL-6 actions, because CaR agonists did not enhance the expression of IL-6 in these cells (Fig. 5).
T. Yamaguchi et al. / Biochemical and Biophysical Research Communications 299 (2002) 532–538
Of note, we and others have shown that CaR agonists stimulated the proliferation of osteoblasts [36], monocytes–macrophages [37], bone marrow stromal cells [38], and fibroblasts [39]. Thus, the present findings that the CaR activation evokes a mitogenic effect on myeloma cells further support the notion that the CaR is involved in this physiological process in a variety of cell types. Elevated expression of the CaR may contribute to the constitutive action of growth signaling cascades, including mitogen-activated protein kinase (MAPK) ones [39,40]. We always observe some basal or constitutive activity of MAPK or other signal pathways in our cells [41–43]. This is how myeloma cells survive in bone marrow microenvironment and the CaR, at least in part, might mediate these survival signals. Taken together, it is likely that myeloma cells in the vicinity of activated osteoclasts within the microenvironment of destructive bone lesions are exposed to a high concentration of Ca2þ o , which is released from resorbed bone. The data presented in this study suggest that myeloma cells could be stimulated to proliferate under such circumstances, by sensing high Ca2þ o through the CaR on their surfaces. Thus the CaR in myeloma cells could participate in a vicious cycle by expanding myeloma cell mass in destructive bone lesions.
Acknowledgments This research was generously supported by Grant 13671156 from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from Mitsui Sumitomo Insurance Welfare Foundation, Japan.
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The extracellular calcium (Ca2o+)-sensing receptor is expressed in myeloma cells and modulates cell proliferation Toru Yamaguchi,a,* Mika Yamauchi,b Toshitsugu Sugimoto,b Dharminder Chauhan,c Kenneth C. Anderson,c Edward M. Brown,d and Kazuo Chiharab a Department of Internal Medicine, Takatsuki General Hospital, 1-3-13 Kosobe-cho, Takatsuki 569-1192, Japan Division of Endocrinology/Metabolism, Neurology, and Hematology/Oncology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan c Division of Hematologic Malignancies, Department of Medicine, Dana-Farber Cancer Institute, Boston, MA, USA Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA b
d
Received 25 October 2002
Abstract The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2þ o ) homeostasis by enabling parathyroid, kidney, and other cells to directly ‘‘sense’’ changes in Ca2þ . In multiple myeloma-associated o bone disease, myeloma cells could raise the level of Ca2þ o within their immediate vicinity in the bone marrow microenvironment, through their known capacity to cause bone destruction by stimulating osteoclastic bone resorption. Thus if myeloma cells expressed the CaR, they might sense these locally elevated levels of Ca2þ o , which could, in turn, potentially modify their function(s) in ways that could contribute to myeloma bone disease or other aspects of the pathophysiology of this disabling hematological malignancy. In this study, we examined the expression of the CaR in three myeloma cell lines, human U266, IM-9, and RPMI8226 cells. CaR protein was present in all three cell lines as assessed by immunocytochemistry and Western blot analysis using a monoclonal antibody specific for the CaR. Moreover, the use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in the three cell lines. Exposure 3þ to known polycationic agonists of the CaR, including high Ca2þ o (2.5 mM), neomycin, and gadolinium (Gd ) as well as a specific CaR activator, NPS R467, augmented cell proliferation in all three cell lines. RT-PCR revealed that U266 cells, but not IM-9 cells or RPMI8226 cells, expressed interleukin-6 (IL-6), the expression of which was not enhanced by treatments with CaR agonists. Therefore, taken together, our data first document the fact that the myeloma cell lines, U266, IM-9, and RPMI8226, all express CaR protein and mRNA. Moreover, the CaR expressed on myeloma cells could sense the locally high levels of Ca2þ o in the vicinity of sites of osteoclastic bone resorption and stimulate their proliferation in an IL-6-independent manner. These processes may result in promoting further growth of the tumor and aggravating the associated bone disease. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Calcium-sensing receptor; Multiple myeloma; U266 cells; IM-9 cells; RPMI8226 cells; Proliferation
Myeloma bone disease is characterized by bone destruction that can produce intractable bone pain; it is caused by bone destruction stimulated by tumor-derived factors that activate osteoclastic bone resorption [1,2]. In most patients, there are multiple discrete lytic lesions adjacent to the site of deposits or nests of myeloma cells, caused by osteoclasts that accumulate and resorb bone in response to tumor-derived products. In patients with * Corresponding author. Fax: +81-726-82-3834. E-mail address: [email protected] (T. Yamaguchi).
myeloma, macrophage inflammatory protein-1a (MIP1a) produced by myeloma cells, in combination with receptor activator of nuclear factor jB ligand (RANKL) and interleukin-6 (IL-6) that are produced by marrow stromal cells in response to myeloma cells, enhances osteoclast formation through their combined effects on osteoclast precursors [3,4]. Hypercalcemia occurs as a consequence of increased osteoclastic bone resorption in about one-third of patients with advanced disease. The levels of the extracellular calcium concentration (Ca2þ o ) underneath resorbing osteoclasts are known to
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reach levels as high as 8–40 mM [5]. Thus it is likely that myeloma cells close to activated osteoclasts are exposed to locally high levels of Ca2þ o in the microenvironment surrounding destructive bone lesions. If these cells possessed the capacity to sense these changes in Ca2þ o , their functional properties might be modulated in ways that could contribute to the pathophysiology of multiple myeloma and its associated bone disease. One candidate for a Ca2þ o -sensing mechanism that could be expressed on myeloma cells is the Ca2þ o -sensing receptor (CaR), which has been cloned from bovine and human parathyroid gland [6,7], rat kidney [8], and thyroid C-cells [9]. The central, non-redundant role of the CaR in humans has been documented by showing that inactivating and activating mutations of the CaR gene cause inherited hyper- and hypocalcemic disorders [10,11], rendering affected family members inappropriately ÔresistantÕ or Ôsensitive,Õ respectively, to the usual effects of Ca2þ o on parathyroid and renal functions. The 3þ CaR binds its cationic ligands, such as Ca2þ o , Gdo , and neomycin, with EC50 values of 3 mM, 20 lM, and 60 lM, respectively. These agonists produce a G protein-dependent activation of intracellular second messenger pathways, including phospholipase C (PLC), leading to elevations in the levels of inositol trisphosphate (IP3 ) and the cytosolic calcium concentration [6]. In a previous study, we found that a variety of cell types within bone marrow express the CaR [12] that are not directly involved in forming or breaking down bone (e.g., the functions of osteoblasts and osteoclasts, respectively). In the marrow, hematopoietic precursors expressing the CaR would likely experience significant variations in the levels of Ca2þ o to which they are exposed as a result of changes in the prevailing rate of bone turnover within the local bone/bone marrow microenvironment. These cells include red blood cell precursors, megakaryocytes (the precursors of blood platelets), monocytes, and macrophages, all of which express relatively high levels of the CaR, and white blood cells precursors, which express lower levels of the receptor [12]. Since myeloma cells also arise from hematopoietic precursors (e.g., plasma cells), it is possible that the CaR is expressed in myeloma cells and can sense locally high levels of Ca2þ o within osteolytic bone lesions in myeloma patients. In this study, therefore, we used the three human myeloma cell lines, U266, IM-9, and RPMI8226 cells, to examine the expression of the CaR in myeloma cells and to assess its role in regulating their proliferation. Materials and methods Materials. All routine culture media were obtained from Invitrogen Japan K.K. (Tokyo, Japan). All chemicals were of the highest grade available commercially. The CaR activator, NPS R-467 [13], was provided by NPS Pharmaceuticals (Salt Lake City, UT).
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Cell culture. The U266 human multiple myeloma-derived cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). The IM-9 and RPMI8226 human multiple myelomaderived cell lines were obtained from the Japanese Collection of Research Bioresources (Sen-nan, Japan). Each cell line was grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin in 5% CO2 at 37 °C. The medium was changed twice weekly and the cells were subcultured in 75 cm2 culture flasks. For morphological evaluation, cells were removed from the flasks, pelleted by centrifugation, and resuspended in phosphate-buffered saline (PBS). The cell suspension was spread on a glass microscope slide, air-dried, fixed with 4% formaldehyde in PBS for 5 min, and washed with PBS. The slides were stored at 4 °C until assessment for the presence of the CaR as described below. Immunocytochemistry for CaR in U266, IM-9, and RPMI8226 cells. A mouse anti-CaR monoclonal antibody ADD was provided by NPS Pharmaceuticals, Salt Lake City, UT. This antibody was raised against a peptide (ADD) corresponding to amino acids 214–235 of the human CaR, which resides within the predicted aminoterminal extracellular domain of the receptor. The antibody was used for immunocytochemistry and Western blot analysis as described below. Fixed U266, IM-9, and RPMI8226 cells were treated with DAKO Protein Block Serum-Free Solution (DAKO Corp.) for 1 h and then incubated overnight at 4 °C with the anti-CaR antibody (ADD) at a concentration of 5 lg/ml in blocking solution. Negative controls were carried out by incubating the cells with the anti-CaR antibody (5 lg/ml) that had been preabsorbed with 10 lg/ml of the ADD peptide. After washing the cells three times with 0.5% bovine serum albumin in PBS for 10 min each, alkaline phosphatase-coupled, goat anti-mouse IgG (1:200; Sigma Chemical, St. Louis, MO) was added and incubated for 1 h at room temperature. The cells were then washed with PBS three times for 10 min each and the color reaction was developed for 10–20 min using a solution consisting of 44 ll nitroblue tetrazolium chloride (NBT) (75 mg/ml) and 33 ll 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) (50 mg/ml) in 10 ml of 0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2 , and 1 mg/ml levamisole, which was included to inhibit endogenous cellular alkaline phosphatase (ALP) activity. The color reaction was stopped by washing the slides twice in the above solution without NBT or BCIP and then twice in water. Western blot analysis of CaR in U266, IM-9, and RPMI8226 cells. U266, IM-9, and RPMI8226 cells in 75 cm2 culture flasks were removed from the flasks, pelleted by centrifugation, resuspended in 1 ml of a lysis solution (1% SDS, 10 mM Tris–HCl, pH 7.4), and heated to 65 °C. The cells were homogenized by brief sonication and heated for an additional 5 min at 65 °C. Insoluble material was removed by centrifugation for 5 min. The resultant whole cell lysate in the supernatant was stored at )20 °C until Western blot analysis was carried out. Aliquots of 75 lg of protein were dissolved in SDS–Laemmli gel loading buffer containing 100 mM dithiothreitol, incubated at 37 °C for 15 min, and resolved electrophoretically on 6.5% SDS–polyacrylamide gels. Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon Transfer; Nihon Millipore, Tokyo, Japan) at 240 mA for 40 min in transfer buffer containing 19 mM Tris– HCl, 150 mM glycine, 0.015% SDS, and 20% methanol. The blots were then blocked with 1% BSA in PBS containing 0.25% Triton X-100 (blocking solution) for 2 h and incubated with ADD or with peptideblocked antibody [the same amount of antibody preincubated at room temperature for 60 min with twice the amount (wt/wt) of the synthetic CaR peptide against which it was raised] at a concentration of 1 lg/ml in the blocking solution overnight at 4 °C. The blots were then washed three times with PBS containing 0.25% Triton X-100 (washing solution) at room temperature for 10 min each. The blots were further incubated with a 1:2000 dilution of ALP-coupled, goat anti-mouse IgG (Sigma Chemical Co.) in the blocking solution for 1 h at room temperature. The blots were then washed three times with washing solution at room temperature for 10 min each and specific protein bands were detected using NBT and BCIP as described above.
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PCR amplification of CaR transcript(s) in U266, IM-9, and RPMI8226 cells. Total RNA was prepared from U266, IM-9, and RPMI8226 cells with the TRIzol Reagent (Invitrogen). Total RNA (1 lg) was used for the synthesis of single-stranded cDNA (cDNA synthesis kit, Invitrogen). The resultant first-stranded cDNA was used for the PCR procedure. PCR was performed at a final concentration of 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl2, 0.2 mM dNTP, 0.4 lM of forward primer, 0.4 lM of reverse primer, and 1 ll of ELONGASE enzyme mix (a Taq/Pyrococcus species GB-D DNA polymerase mixture) (Invitrogen). The primer sequences used were sense primer, 50 -AAGCACCTACGGCATCTAA-30 (nucleotides 1384–1402 of the human CaR) and antisense primer, 50 -GCGATCCC AAAGGGCTCCG-30 (nucleotides 1826–1844), according to the method of Goebel et al. [14]. These were intron-spanning primers to avoid confusion arising from amplification of the same sequences within genomic DNA. The PCR for the human CaR was run under the following conditions: initial denaturation at 94 °C for 5 min, 38 cycles of amplification (50-s denaturation at 94 °C, 50-s annealing at 50 °C, and 2-min extension at 72 °C). The reaction was completed with an additional 8-min incubation at 72 °C to allow completion of extension. PCR products were fractionated on 1.2% agarose gels. The presence of a 461 nucleotide-long amplified product was indicative of a positive PCR arising from cDNA. The PCR product in the reaction mixture was purified using the QIAquick PCR purification kit (Qiagen, Santa Clarita, CA) and subjected to direct, bi-directional sequencing employing the same primer pairs used for PCR by means of an automated sequencer (AB377, Applied Biosystems, Foster City, CA) using dideoxy terminator Taq technology. Assays of cell proliferation. Proliferation of myeloma cells was assessed by counting cell numbers using a hemocytometer or by a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay is a colorimetric assay used to determine cell number and/or assess cellular viability, based on the ability of mitochondria in live cells to oxidize thiazolyl blue, a tetrazolium salt. One hundred ll each of the U266, IM-9, and RPMI8226 cell lines in the growth media was seeded in 96-well plates at a density of 1 104 cells/ml. After incubating for appropriate periods and removing medium for counting cell numbers, 10 ll MTT (10.4 mg/ml) was added to each well containing 40 ll of myeloma cells. The plates were incubated at 37 °C in humidified 5% CO2 for 4 h to convert water-soluble MTT to insoluble formazan, which occurs only in viable cells. After 4 h, 100 ll of 50% dimethylformamide with 20% SDS was added to each well to solubilize formazan and the plates were incubated at 37 °C overnight. Absorbance was then measured at 595 nm with background subtraction at 655 nm. PCR amplification of IL-6 in U266, IM-9, and RPMI8226 cells. U266, IM-9, and RPMI8226 cells were incubated with serum-free PRPMI1640 medium for 24 h in the absence or presence of Ca2þ 2.5 mM or NPS R467 1 lM. After the preparation of total RNA and the synthesis of singlestranded cDNA, the PCR was performed as described above. The primer sequences used for human IL-6 were sense primer, 50 -CATCCTCGACG GCATCTCAG-30 and antisense primer, 50 -GTCCTAACGCTCATA CTTTT-30 . The PCR was run by 27 cycles of amplification (30-s denaturation at 94 °C, 30-s annealing at 52 °C, and 1-min extension at 72 °C) and an additional 10-min incubation at 72 °C. PCR products were fractionated on 1.2% agarose gels. The presence of a 582 nucleotide-long amplified product was indicative of a positive PCR arising from cDNA. The PCR for GAPDH was also performed as a control.
Results Immunoreactivity of CaR protein in U266, IM-9, and RPMI8226 cells using CaR-specific antibody To clarify whether the CaR is expressed in myeloma cells, we investigated the presence of the receptor in
Fig. 1. Immunocytochemistry of U266, IM-9, and RPMI8226 cells carried out as described in Materials and methods using a monoclonal CaR-specific antibody (ADD). Immunocytochemistry revealed moderate to strong CaR staining in the three cells (upper panel), which were eliminated by preincubating the primary antibody with the peptide against which it was raised (lower panel). The photomicrographs were taken at a magnification of 400.
three myeloma cell lines, U266, RPMI8226, and IM-9. Immunocytochemistry of these cells with a CaR-specific monoclonal antibody revealed moderate to strong CaR staining (Fig. 1, upper panel), which was eliminated by preincubating the primary antibody with the peptide against which it was raised (Fig. 1, lower panel). We also performed Western blot analysis on proteins isolated from U266, RPMI8226, and IM-9 cells (Fig. 2, left). In proteins in total cellular lysates obtained from the three cell lines, the ADD stained double bands at
Fig. 2. Left: Western blot analysis of whole cell lysates from U266, IM9, and RPMI8226 cells performed as described in Materials and methods. Double bands at molecular weights of 160 kDa that were stained in the presence of specific antibody were consistent with the intact, glycosylated CaR. The specificity of the labeling by the antiCaR antibody used in this study was confirmed by reduction or abolition of the bands in extracts of these cells when they were incubated with antibody preabsorbed with the peptide against which it was raised. Right: Analysis of CaR transcripts in U266, IM-9, and RPMI8226 cells by RT-PCR, performed as described in Materials and methods. The product was of the expected size, 461 bp, for a product derived from authentic CaR transcript(s). No products were observed when the reverse transcriptase (RT) was omitted during synthesis of cDNA.
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molecular weights of 160 kDa-sizes consistent with the intact glycosylated human CaR [15,16]. These bands were specific, since it was markedly diminished in intensity following preabsorption of the primary antibody with its specific peptide. Detection of CaR mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR RT-PCR with CaR-specific, intron-spanning primers, which were utilized to ensure that the CaR products was not the result of amplification of contaminating genomic DNA segments, amplified products of the expected size from U266, IM-9, and RPMI8226 cells, 461 bp (Fig. 2, right). No products were observed when the reverse transcriptase (RT) was omitted during synthesis of cDNA. DNA sequence analysis of the PCR products revealed 100% identities with the corresponding region of the human parathyroid CaR cDNA sequences [7] (not shown). These results show that the RT-PCR
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product corresponded to an authentic CaR sequence, indicating the presence of bona fide CaR transcript(s) in these cells. The effects of CaR agonists on the proliferation of U266, IM-9, and RPMI8226 cells When cell numbers were counted, high Ca2þ o (2.5 mM), neomycin sulfate (100 lM) and Gd3þ o (25 lM), polycationic agonists of the CaR, as well as NPS R467 (1 lM), a specific CaR activator, significantly stimulated the proliferation of U266, IM-9, and RPMI8226 cells, up to treatments for 3 days (Fig. 3) (p < 0:05). The MTT assay performed after treatments for 1 day also showed that the CaR agonists significantly stimulated the proliferation of the three cells (p < 0:05) (Fig. 4). The effects of CaR agonists on the expression of IL-6 mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR We performed RT-PCR for IL-6 in U266, IM-9, and RPMI8226 cells in order to examine whether or not CaR agonists exert their mitogenic effect on the cells by enhancing the expression of endogenous IL-6 in the cell
Fig. 3. Effects of CaR-agonists on the proliferation of U266, IM-9, and RPMI8226 cells. Cell numbers were counted up to 3 days under treatments with various CaR-agonists as described in Materials and methods. Each bar represents the mean SEM for nine determinations. * p < 0:05 compared to cells treated with 0.5 mM Ca2þ o .
Fig. 4. Effects of CaR-agonists on the proliferation of U266, IM-9, and RPMI8226 cells. The MTT assay was performed after 1 day treatments of the cells with CaR-agonists as described in Materials and methods. Each bar represents the mean SEM for eight determinations. * p < 0:05 compared to cells treated with 0.5 mM Ca2þ o .
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Fig. 5. The effects of CaR agonists on the expression of IL-6 mRNA in U266, IM-9, and RPMI8226 cells by RT-PCR, performed as described in Materials and methods. U266 cells expressed IL-6 under a basal Ca2þ condition (0.5 mM), but the expression was not enhanced by o treatments with high Ca2þ o (2.5 mM) or NPS R467 (1 lM). IL-6 was not expressed in IM-9 cells or RPMI8226 cells under either basal Ca2þ o or CaR agonist-treated conditions.
lines (Fig. 5). Although U266 cells expressed IL-6 under a basal Ca2þ o condition (0.5 mM), the expression was not augmented by the treatment with high Ca2þ o (2.5 mM) or NPS R467 (1 lM). IL-6 was not expressed in IM-9 cells or RPMI8226 cells under either basal Ca2þ o or CaR agonist-treated conditions.
Discussion Our results showed that U266, IM-9, and RPMI8226 cell lines clearly expressed CaR protein by immunocytochemistry (Fig. 1) as well as by Western blot analysis (Fig. 2, left), which revealed specific double bands at molecular weights consistent with those of the intact, glycosylated CaR [15,16]. In addition, RT-PCR performed on total RNA isolated from these three cell lines followed by sequence analysis of the PCR products indicated the presence of bona fide CaR transcripts (Fig. 2, right). Thus, the present study shows that these myeloma cell lines express both CaR protein and mRNA and the receptor could potentially sense local changes in Ca2þ related to excessive osteoclastic bone resorption o associated with myeloma bone disease. The functions of the CaR in hematopoietic cells, including myeloma cells, are not well understood. There is
a limited body of data, however, indicating that changes have direct actions on these CaR-expressing in Ca2þ o hematopoietic elements that are of potential physiological relevance. Elevated levels of Ca2þ o have several effects on macrophages or monocytes. First, high Ca2þ o promotes chemotaxis of peripheral blood monocytes [17]; therefore, Ca2þ and the CaR share some of the o properties of chemokines and chemokine receptors, respectively [18]. Second, Bornefalk et al. [19] have shown that high Ca2þ o stimulates the secretion of IL-6 both in vivo and in vitro from peripheral blood monocytes. Finally, raising Ca2þ o potentiates the fusion of rat alveolar macrophages induced by 1,25-dihydroxyvitamin D3 [20]. Elevating Ca2þ also stimulates the formation of o erythroid colonies in vitro and raises the cytosolic calcium concentration in erythroid precursors isolated from uremic patients, an effect that is potentiated by 1,25ðOHÞ2 D3 [21], which is known to upregulate CaR expression [22]. Ca2þ o also modulates several processes in platelets, including stimulating the release of arachidonic acid [23] and inhibiting the accumulation of cAMP [24]—effects that might potentially be CaR-mediated. Interestingly, Ca2þ o has been quantified directly in platelet clumps during platelet activation and it decreases to a substantial extent [25]. Thus while much additional work needs to be carried out defining CaRÕs potential roles in cells of various hematopoietic lineages, available data suggest that (a) several of these cells express the CaR, (b) Ca2þ o modulates their functions, and (c) Ca2þ can change within their local microenvirono ments—either within the bone marrow as a function of bone turnover or within peripheral blood (e.g., within clumps of activated platelets). IL-6 is proposed to be either an autocrine or paracrine growth factor for some human myeloma cells [26– 28]. Kawano et al. [28] have postulated an autocrine growth mechanism because myeloma cells secrete IL-6 and proliferate in response to this cytokine in vitro. More recently, myeloma cells have been triggered via their cell surface CD40 to both secrete IL-6 and to proliferate [29,30], suggesting the potential for induction of IL-6-mediated autocrine growth in myeloma. The possible role of IL-6-mediated paracrine control of myeloma cell growth is supported by observations that (a) bone marrow stromal cells are the major source of IL-6 in myeloma [27,31,32], (b) freshly isolated myeloma cells cultured without exogenous IL-6 rapidly stop proliferating [33], and (c) adhesion of myeloma cells to stromal cells up-regulates IL-6 secretion by stromal cells [34,35]. In this study, we found that CaR agonists stimulated proliferation of U266, IM-9, and RPMI8226 cells (Figs. 3 and 4). However, the mechanisms by which CaR agonists exert a mitogenic effect on myeloma cells seemed to be independent to IL-6 actions, because CaR agonists did not enhance the expression of IL-6 in these cells (Fig. 5).
T. Yamaguchi et al. / Biochemical and Biophysical Research Communications 299 (2002) 532–538
Of note, we and others have shown that CaR agonists stimulated the proliferation of osteoblasts [36], monocytes–macrophages [37], bone marrow stromal cells [38], and fibroblasts [39]. Thus, the present findings that the CaR activation evokes a mitogenic effect on myeloma cells further support the notion that the CaR is involved in this physiological process in a variety of cell types. Elevated expression of the CaR may contribute to the constitutive action of growth signaling cascades, including mitogen-activated protein kinase (MAPK) ones [39,40]. We always observe some basal or constitutive activity of MAPK or other signal pathways in our cells [41–43]. This is how myeloma cells survive in bone marrow microenvironment and the CaR, at least in part, might mediate these survival signals. Taken together, it is likely that myeloma cells in the vicinity of activated osteoclasts within the microenvironment of destructive bone lesions are exposed to a high concentration of Ca2þ o , which is released from resorbed bone. The data presented in this study suggest that myeloma cells could be stimulated to proliferate under such circumstances, by sensing high Ca2þ o through the CaR on their surfaces. Thus the CaR in myeloma cells could participate in a vicious cycle by expanding myeloma cell mass in destructive bone lesions.
Acknowledgments This research was generously supported by Grant 13671156 from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from Mitsui Sumitomo Insurance Welfare Foundation, Japan.
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