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Biochemical and Biophysical Research Communications 279, 363–368 (2000)

doi:10.1006/bbrc.2000.3955, available online at http://www.idealibrary.com on

inases by Extracellular Calcium-Sensing Receptorgonists Induces Mitogenic Responses in theouse Osteoblastic MC3T3-E1 Cell Line

oru Yamaguchi, Naibedya Chattopadhyay, Olga Kifor,ennifer L. Sanders, and Edward M. Brown1

ndocrine-Hypertension Division and Membrane Biology Program, Department of Medicine,righam and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

eceived November 10, 2000

Key Words: calcium-sensing receptor; osteoblast;p

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Recently, substantial evidence has accumulatedhat the G-protein-coupled, extracellular calciumCao

21)-sensing receptor (CaR) is expressed in bonearrow-derived cells, including osteoblasts, stromal

ells, monocytes–macrophages, and osteoclast precur-or cells. Our previous studies have shown that theouse osteoblastic MC3T3-E1 cell line also expresses

he CaR and exhibits mitogenic responses when ex-osed to various CaR agonists. In this study, in ordero understand the signaling pathway(s) mediating thisesponse, we studied the effects of CaR agonists onhe phosphorylation of p42/44 mitogen-activated pro-ein kinase (MAPK) (Erk1/2), p38 MAPK, and c-Jun-terminal kinase (JNK) in MC3T3-E1 cells. Raising

he level of Cao21 (4.5 mM) or addition of the poly-

ationic CaR agonists, gadolinium (Gd31) (25 mM), neo-ycin (300 mM) or spermine (1 mM), each stimulated

hosphorylation of both p42/44 and p38 MAPKs, butot JNK, as assessed using phospho-specific antibod-

es to the respective MAPKs. Furthermore, phosphor-lation of p42/44 and p38 MAPK were markedly inhib-ted by their selective and potent inhibitors, PD9805950 mM) and SB203580 (10 mM), respectively. Finally,he two inhibitors suppressed [3H]thymidine incorpo-ation into DNA in MC3T3-E1 cells at a normal level ofao

21 (1.8 mM) as well as when stimulated by high (4.5M) Cao

21, Gd31, or neomycin. Thus, in mouse osteo-lastic MC3T3-E1 cells, both the p42/44 and p38 MAPKascades play pivotal roles in CaR-stimulated mito-enic responses. © 2000 Academic Press

1 To whom correspondence should be addressed at Endocrine-ypertension Division, Brigham and Women’s Hospital, 221 Long-ood Avenue, Boston, MA 02115. Fax: 1-617-732-5764. E-mail:mbrown@rics.bwh.harvard.edu.

363

roliferation; MAPK; p42/44, p38

Bone formation during the normal process of skele-al remodeling is initiated by the migration ofacrophage-like mononuclear cells and preosteoblasts

o sites of osteoclastic bone resorption during the “re-ersal” phase of skeletal turnover that precedes theormation of new bone (1). We have previously demon-trated the expression of the G-protein-coupled, extra-ellular calcium (Cao

21)-sensing receptor (CaR) (2, 3) iniverse cell types in human bone marrow (4), in thesteoblast-like cell lines, UMR-106 and SAOS-2 (5),nd in human peripheral blood monocytes (6). Re-ently, we also found that three different murine cellines—J774 monocyte–macrophage-like cells (7), ST2one marrow-derived stromal cells (8), and theC3T3-E1 osteoblastic cell line (9)—all express theaR. Moreover, CaR agonists stimulate chemotaxisnd proliferation of these three cell lines (7–9), sug-esting that the CaR could potentially represent theolecular mediator of the actions of high Cao

21 onhese two physiological responses. In addition, Kana-ani et al. have reported that osteoclast precursorsxpress the CaR and that CaR agonists suppress theormation of mature osteoclasts from their precursorells as well as their bone resorptive activity (10).aken together, these findings suggest an importantole for the CaR in the key “reversal” phase of boneemodeling. By sensing Cao

21 released by osteoclast-ediated bone resorption, the CaR may contribute to

he suppression of osteoclastic bone resorption as wells the chemotaxis and proliferation of cell populations

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

needed for the orderly transition from bone breakdownt

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Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o its subsequent replacement by osteoblasts.Other researchers have also reported that the CaR is

xpressed in osteoblastic MC3T3-E1 cells (10–12) andhat high Cao

21 and other CaR agonists, including gad-linium (Gd31) and neomycin, stimulate chemotaxisnd proliferation of these cells (13, 14). The CaR isnown to bind its agonists and produce a G-protein-ependent activation of phospholipase C (PLC), lead-ng to an elevation in the cellular levels of inositolrisphosphate (IP3), thereby releasing Ca21 from itsntracellular stores and producing transient increasesn the cytosolic calcium concentration (Cai

21). Godwinnd Soltoff have shown that inhibiting the activation ofither G-protein or PLC blocks nearly all of theigh Cao

21-stimulated chemotaxis of MC3T3-E1 cells,hereas blocking protein kinase C (PKC) or phos-hoinositide-3-kinase did not (14). These resultsuggested that Cao

21-stimulated chemotaxis of this celline is linked to the activation of G-protein and PLC. Inontrast, another study reported that neither Gd31 norigh Cao

21 increased either inositol phosphate forma-ion or Cai

21 in MC3T3-E1 cells (15). A third studyeported that an increase in Cao

21 caused only a gradualnd small increase in Cai

21 in these cells and thatantrolene—an inhibitor of calcium release from itsntracellular stores—had no effect on high Cao

21-licited stimulation of DNA synthesis (16). Thus theignaling pathways by which Cao

21 or other CaR ago-ists mediate their mitogenic actions in MC3T3-E1ells remain unclear, and it seems unlikely that inosi-ol phosphate formation and resultant mobilization ofntracellular Ca21 participate importantly in this pro-ess.

A family of mitogen-activated protein kinasesMAPK) has recently been characterized in mamma-ian cells, which are activated by dual phosphorylationn threonine and tyrosine residues. p42/44 MAPKErk1/2) participate in mitogenic responses to growthactors (17, 18). p38 MAPK and c-Jun N-terminal ki-ase (JNK) are two additional MAPK family membershat are activated by a variety of cellular stresses,ncluding osmotic shock, inflammatory cytokines, lipo-olysaccharides, UV light and growth factors (19, 20).ecent evidence indicates that p38 MAPK also plays aajor role in the mitogenic responses of T cells to

nterleukin-2 (21) and of hepatocytes to catechol-mines during the regeneration of the liver that followsartial hepatectomy (22).Recently, Matsuda et al. have reported that theseAPK cascades modulate the proliferation and differ-

ntiation of human osteoblasts in response to epider-al growth factor, hypoxia, and mechanical stress (23).hus it is possible that the mitogenic responses ofsteoblastic MC3T3-E1 cells to CaR agonists are alsoediated by these MAPK cascades. We have now ex-

364

amilies of MAPKs and, in turn, DNA synthesis inC3T3-E1 cells using Western blotting with phos-

hospecific antibodies to the various MAPKs, selectivenhibitors of p42/44 and p38 MAPK (24) and measure-

ents of [3H]-thymidine incorporation into DNA. Ouresults indicate that both p42/44 and p38 MAPKs con-ribute to CaR agonist-induced mitogenic responses inhis cell line.

ATERIALS AND METHODS

Materials. All routine cell culture media were obtained fromIBCO/BRL (Grand Island, NY). Neomycin sulfate and anhydrous

alcium chloride (CaCl2) were purchased from Sigma Chemical Co.St. Louis, MO), and Gd31 (III) chloride hexahydrate (GdCl3 z 6H2O)as from Aldrich Chemical Co. (Milwaukee, WI). [3H]-ethylthymidine was purchased from DuPont-New England Nu-

lear (Boston, MA). PD98059 and SB203580 were obtained fromalbiochem-Novabiochem (Nottingham, UK). All other chemicalsere of the highest grade commercially available.

Cell culture. MC3T3-E1 cells—established as an osteoblastic celline from normal mouse calvaria (25, 26)—were a generous gift fromr. H. Kodama (Ohu University, Koriyama, Japan). MC3T3-E1 cellsere grown in a-Modified Minimal Essential Medium (a-MEM; Ca21,.8 mM; Mg21, 0.81 mM; H2PO4, 1.0 mM) supplemented with 10%etal bovine serum (FBS) (Hyclone, Logan, UT) and 1% penicillin/treptomycin in 5% CO2 at 37°C. The medium was changed twiceeekly, and the cells were subcultured into 25-cm2 culture flasks byetaching them gently with a cell scraper after they reached subcon-uency.

Western blot analysis for phosphorylated MAPK. Rabbit poly-lonal antibodies specific for the phosphorylated forms of p42/44APK (Thr202/Tyr204), p38 MAPK (Thr180/Tyr182), and JNK

Thr183/Tyr185) were purchased from New England Biolabs, Inc.Beverly, MA). Preparation of cellular extracts and Western blotnalyses were carried out according to the manufacturer’s recom-endations. Subconfluent MC3T3-E1 cells in 6-well plates were

ncubated overnight with a-MEM containing 0.5% bovine serumlbumin (BSA) in the presence or absence of PD98059 orB203580. The cells were then treated with CaR agonists in theresence or absence of the same inhibitor in fresh medium with.5% BSA for up to 60 min as indicated in the figures and Results.s a control, cells were also treated with 1027 M phorbol myristatecetate (PMA) for 15 min. At the end of the experiments the cellsere lysed with 100 ml of SDS sample buffer containing 50 mMithiothreitol after being washed once with phosphate-bufferedaline (PBS). The cell lysates were then scraped from the plates,ransferred to microcentrifuge tubes and heated for 5 min at00°C. The viscosity of the samples was reduced by brief sonica-ion, and 20 ml of each of the resultant whole cell lysates wasubjected to SDS–polyacrylamide gel electrophoresis on 7.5%DS–polyacrylamide gels. Proteins were subsequently trans-

erred electrophoretically to nitrocellulose blots at 240 mA for 40in in transfer buffer containing 19 mM Tris–HCl, 150 mM gly-

ine, 0.015% SDS and 20% methanol. The blots were blocked forh with 1% BSA in PBS containing 0.25% Triton X-100 (blocking

olution) and then incubated overnight at 4°C with the respectiventibodies. The blots were washed three times with PBS contain-ng 0.25% Triton X-100 (washing solution) at room temperatureor 10 min each. The blots were further incubated with a 1:2000ilution of horseradish peroxidase-coupled, goat anti-rabbit IgGSigma Chemical Co., St. Louis, MO) in blocking solution for 1 h atoom temperature. The blots were then washed three times with

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Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

he washing solution at room temperature for 40 min each, andpecific protein bands were detected using enhanced chemilumi-escence (ECL) (Amersham, Arlington Heights, IL).

DNA synthesis in MC3T3-E1 cells. We assessed DNA synthesisn MC3T3-E1 cells using [3H]-thymidine incorporation. MC3T3-E1ells were removed from culture plates by gentle scraping and re-eated pipetting and seeded in 24-well plates at a density of 1000ells/well in 500 ml of a-MEM containing 10% FBS as well as variousoncentrations of Cao

21, neomycin, or Gd31 in the presence or absencef PD98059 or SB203580 as indicated in the figures. After a 48 hncubation at 37°C, cells were pulsed with [3H]-thymidine (1 mCi/ell). Incubations were terminated after an additional overnight

ncubation by removal of the medium and addition of 5% trichloro-cetic acid (TCA). Cells were subsequently scraped and transferredo microcentrifuge tubes. After centrifugation at 15,000g and re-oval of the supernatant, the precipitate was washed with 75%

thanol and desiccated at room temperature. The residual pellet wasissolved in 20 mM NaOH and 1% SDS, and a scintillation cocktailas added. Samples were counted in a liquid scintillation counter.

Statistics. Results are expressed as the mean 6 SEM. Statisticalvaluations for differences between groups were carried using one-ay analysis of variance (ANOVA) followed by Fisher’s protected

east significant difference (PLSD). For all statistical tests, a value of, 0.05 was considered to indicate a statistically significant dif-

erence.

ESULTS AND DISCUSSION

To determine whether or not the CaR is linked toAPK cascades in mouse osteoblastic MC3T3-E1 cells,e examined the effects of various CaR agonists on thehosphorylation of p42/44 MAPK, p38 MAPK and JNKy Western blot analyses using antibodies specific forhe phosphorylated form(s) of each MAPK (Figs. 1 and). Treatment of the cells with high Cao

21 (4.5 mM)timulated phosphorylation of both the p42/44 and p38APKs, while low Cao

21 (0.5 mM) had no effect onither of these MAPKs (Fig. 1). Incubation of the cellsith Gd31 (25 mM), neomycin (300 mM) or spermine (1M) also stimulated phosphorylation of both the

42/44 and p38 MAPKs (Fig. 2). Maximal increases in

FIG. 1. Effects of exposure to low or high Cao21 on the phosphor-

lation of p42/44 MAPK, p38 MAPK, and JNK in mouse osteoblasticC3T3-E1 cells. Subconfluent cells in a-MEM containing 0.5% BSAere exposed to low (0.5 mM) or high (4.5 mM) Cao

21 for up to 30 min,nd then whole cell lysates were subjected to Western blot analysissing antibodies specific for the phosphorylated forms of each of theAPKs.

365

igh Cao21, 30 min following addition of Gd31 and neo-

ycin, and 7 min after adding spermine. In contrast,reatment with high Cao

21 had no effect on the phos-horylation of JNK at any of the time points examinedp to 30 min (Fig. 1).Overnight pretreatment of the cells with PD98059

50 mM) or SB203580 (10 mM) specifically and mark-dly suppressed the high Cao

21-evoked phosphoryla-ions of the p42/44 and p38 MAPKs, respectively (Fig.). These findings show that CaR activation is linked tohosphorylation of both p42/44 and p38 MAPKs inC3T3-E1 cells, and document that PD98059 and

B203580 act as selective and potent inhibitors of the42/44 and p38 MAPK cascades, respectively, whenhey are activated by CaR agonists in these cells.

Next, to study whether the CaR agonist-inducedtimulation of MAPK phosphorylation are linked toitogenic responses in MC3T3-E1 cells, we examined

he effects of PD98059 and SB203580 on DNA synthe-is stimulated by these agents in MC3T3-E1 cells. Ashown in Fig. 4A, treatment of the cells with PD9805950 mM) or SB203580 (10 mM) significantly suppressed3H]-thymidine incorporation at a physiological level ofao

21 (1.8 mM) (P , 0.05), while lower concentrationsf either PD98059 (10 mM) or SB203580 (1 mM) had noignificant effects on DNA synthesis. The stimulationf [3H]-thymidine incorporation by high Cao

21 (4.8 mM)as also significantly suppressed by PD98059 (10 and0 mM) or SB203580 (10 mM) (P , 0.05). The concen-ration dependence of these inhibitory effects of

FIG. 2. Effects of various CaR agonists on the phosphorylation of42/44 MAPK and p38 MAPKs in MC3T3-E1 cells. Subconfluentells in a-MEM containing 0.5% BSA were exposed to the variousolycationic CaR agonists indicated in the figure for up to 60 min,nd then whole cell lysates were subjected to Western blot analysissing antibodies specific for the phosphorylated forms of the p42/4nd p38 MAPKs.

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Vol. 279, No. 2, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

D98059 in MC3T3-E1 cells are similar to those re-orted previously in vascular smooth muscle cells, inhich angiotensin II-stimulated [3H]-thymidine incor-oration was inhibited by PD98059 with an IC50 of7.8 6 1.6 mM (27).Treatment of the cells with PD98059 (50 mM) or

B203580 (10 mM) also significantly suppressed thetimulation of [3H]-thymidine incorporation by otheraR agonists, including Gd31 (25 mM) and neomycin

300 mM) (P , 0.05) (Fig. 4B). These findings, togetherith those shown in Fig. 3, suggest that both the42/44 and p38 MAPK cascades are important media-ors of the mitogenic actions of the CaR in MC3T3-E1ells, although the former may be more important thanhe latter in this regard.

Although several studies have shown that the CaR isxpressed in various osteoblastic cells, including theurine osteoblastic MC3T3-E1 cell line (5, 9–11),

hese earlier investigations provided no definitive evi-ence that the receptor is linked to any physiologicalesponses in these cells. However, we recently showedhat the CaR mediates the opening of an outward K1

hannel in MC3T3-E1 cells in studies utilizing thepecific CaR activator, NPS R467 (28), thereby docu-enting that this receptor is not only expressed in but

s functionally active in this cell line. The present studyrovides additional evidence that the CaR is function-lly linked to the p42/44 and p38 MAPK cascades andhereby induces mitogenic responses in MC3T3-E1ells.

In summary, therefore, both the p42/44 and p38APK cascades play pivotal roles in CaR-stimulated

FIG. 3. Effects of high Cao21 on the activities of the p42/44 and

38 MAPKs in MC3T3-E1 cells in the presence or absence of theAPK inhibitors, PD98059 or SB203580. Subconfluent cells were

ncubated overnight in a-MEM containing 0.5% BSA in the presencer absence of the specific p42/44 and p38 MAPK inhibitors, PD9805950 mM) and SB203580 (10 mM), respectively. The cells were thenreated with high Cao

21 (4.5 mM) in the presence or absence of theame inhibitors in fresh media with 0.5% BSA, for up to 30 min asndicated in the figure.

366

FIG. 4. (A) Effects of treatment with PD98059 or SB203580 onasal and high Cao

21-stimulated DNA synthesis in MC3T3-E1ells. The cells were treated with normal Cao

21 (1.8 mM) or highao

21 (4.8 mM) in the presence or absence of PD98059 (10 and 50M) or SB203580 (1 and 10 mM) for 3 days. [3H]Thymidine incor-oration was then measured as described under Materials andethods. (B) Effects of treatment with PD98059 or SB203580 onNA synthesis in MC3T3-E1 cells stimulated by various CaRgonists. MC3T3-E1 cells were treated with Gd31 or neomycin inhe presence or absence of PD98059 (50 mM) or SB203580 (10 mM)or 3 days and then pulsed with [3H]thymidine as described under

aterials and Methods. Each bar represents the mean 6 SEM forix determinations. *P , 0.05 compared to cells treated with.8 mM Cao

21 alone. **P , 0.05 compared to cells treated with.8 mM Cao

21 alone or 1.8 mM Cao21 plus the indicated CaR

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ells. These responses could, in turn, be part of theechanism through which osteoclastic bone resorption

s coupled to subsequent osteoblastic replacement ofhe missing bone. In other words, local elevations inao

21—by stimulating CaR-mediated mitogenic andhemotactic responses of preosteoblasts residentithin the bone/bone marrow microenvironment—may

erve to ensure the availability of adequate numbers ofsteoblastic cells at sites of recent bone resorption.

CKNOWLEDGMENTS

The authors gratefully acknowledge generous grant support fromhe following sources: USPHS Grants DK41415, DK48330, andK52005, NPS Pharmaceuticals, Inc., the St. Giles Foundation and

he National Space Bioscience Research Institute (NSBRI) (to.M.B.), the Mochida Memorial Foundation Grant for Medical andharmaceutical Research, and the Yamanouchi Foundation Grant

or Research on Metabolic Disorders (to T.Y.).

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