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Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-β/MAPK Signaling Pathways

  • Kerstin Tiedemann
    Affiliations
    Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Osama Hussein
    Footnotes
    Affiliations
    Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Gulzhakhan Sadvakassova
    Affiliations
    Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Yubin Guo
    Affiliations
    Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Peter M. Siegel
    Footnotes
    Affiliations
    Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 2B2, Canada

    Department of Biochemistry, McGill University, Montreal, Quebec H3A 2B2, Canada

    Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Svetlana V. Komarova
    Correspondence
    Holds a Canada Research Chair in osteoclast biology and is supported by operating grants from IMHA/CIHR and the Cancer Research Society. To whom correspondence should be addressed: Faculty of Dentistry, McGill University, 740 Dr. Penfield Ave., Montreal, Quebec H3A 1A4, Canada. Tel.: 514-398-4314; Fax: 514-398-8900
    Affiliations
    Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada

    Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Author Footnotes
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
    2 Holds a research scientist award from the CCSRI and is supported by an operating grant from the CBCRA/Canadian Institutes of Health Research (CIHR).
    1 Supported by Government of Quebec's Merit Doctoral Research Scholarship Program, by Lloyd-Carr Harris Fellowship, and by McGill University.
Open AccessPublished:September 30, 2009DOI:https://doi.org/10.1074/jbc.M109.010785
      Breast cancer commonly metastasizes to bone where its growth depends on the action of bone-resorbing osteoclasts. We have previously shown that breast cancer cells secrete factors able to directly stimulate osteoclastogenesis from receptor activator of nuclear factor κB ligand (RANKL)-primed precursors and that transforming growth factor-β (TGFβ) plays a permissive role in this process. Now, we evaluate the signaling events triggered in osteoclast precursors by soluble factors produced by MDA-MB-231 human breast carcinoma cells. In mouse bone marrow cultures and RAW 264.7 murine monocytic cells, MDA-MB-231-derived factors increased osteoclast number, size, and nucleation. These factors failed to induce Smad2 phosphorylation, and short interfering RNAs against Smad4 did not affect their ability to induce osteoclastogenesis. In contrast, MDA-MB-231 factors induced phosphorylation of p38 and ERK1/2, and pharmacological inhibitors against p38 (SB203580) and MEK1/2 (PD98059) impeded the osteoclastogenic effects of cancer-derived factors. Neutralizing antibodies against TGFβ attenuated p38 activation, whereas activation of ERK1/2 was shortened in duration, but not decreased in amplitude. ERK1/2 phosphorylation induced by cancer-derived factors was blocked by MEK1/2 inhibitor, but not by Ras (manumycin A) or Raf (GW5074) inhibitors. Inhibition of protein kinase Cα using Gö6976 prevented both ERK1/2 phosphorylation and osteoclast formation in response to MDA-MB-231-derived factors. Using microspectrofluorimetry of fura-2-AM-loaded osteoclast precursors, we have found that cancer-derived factors, similar to RANKL, induced sustained oscillations in cytosolic free calcium. The calcium chelator BAPTA prevented calcium elevations and osteoclast formation in response to MDA-MB-231-derived factors. Thus, we have shown that breast cancer-derived factors induce osteoclastogenesis through the activation of calcium/protein kinase Cα and TGFβ-dependent ERK1/2 and p38 signaling pathways.

      Introduction

      Breast cancer exhibits a high propensity to metastasize to bone causing bone pain, pathological fractures, hypercalcemia, spinal cord compression, and immobility (
      • Mundy G.R.
      ,
      • Coleman R.E.
      ). Breast cancer cells do not resorb bone; instead they rely on stimulation of osteoclasts, cells physiologically responsible for bone destruction (
      • Mundy G.R.
      ,
      • Coleman R.E.
      ,
      • Joyce J.A.
      • Pollard J.W.
      ,
      • Futakuchi M.
      • Nannuru K.C.
      • Varney M.L.
      • Sadanandam A.
      • Nakao K.
      • Asai K.
      • Shirai T.
      • Sato S.Y.
      • Singh R.K.
      ). Breast cancer cells can stimulate osteoclasts indirectly, by producing factors, such as parathyroid hormone-related peptide, interleukin-1, -6, and -11, which act on bone-forming osteoblasts to increase the production of an essential osteoclast stimulator, receptor activator of nuclear factor κB (RANK)
      The abbreviations used are: RANK
      receptor activator of nuclear factor κB
      RANKL
      RANK ligand
      BAPTA
      1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, [Ca2+]i, cytosolic free Ca2+ concentration
      CM
      conditioned medium
      JNK
      c-Jun N-terminal kinase
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      MEK
      MAPK/ERK kinase
      NFAT
      nuclear factor of activated T cells
      OC
      osteoclast
      PKC
      protein kinase C
      TβRI
      TGFβ type I receptor
      MKK
      MAPK kinase
      DMEM
      Dulbecco's modified Eagle's medium
      GST
      glutathione S-transferase
      TRAP
      tartrate-resistant acid phosphatase.
      ligand (RANKL) (
      • Mundy G.R.
      ,
      • Fili S.
      • Karalaki M.
      • Schaller B.
      ,
      • Mundy G.R.
      ,
      • Oyajobi B.O.
      • Mundy G.R.
      ,
      • Guise T.A.
      ,
      • Siclari V.A.
      • Guise T.A.
      • Chirgwin J.M.
      ,
      • Asagiri M.
      • Takayanagi H.
      ,
      • Roodman G.D.
      • Dougall W.C.
      ).
      We have found that soluble factors produced by human or mouse breast cancer cells can directly stimulate osteoclast formation from late human or mouse osteoclast precursors (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). These effects depended on the permissive action of TGFβ, and we observed that TGFβ type I receptor expression (TβRI) was up-regulated in late osteoclast precursors (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). The expression of TGFβ and TβRI increases at the interface between tumor and bone in vivo (
      • Futakuchi M.
      • Nannuru K.C.
      • Varney M.L.
      • Sadanandam A.
      • Nakao K.
      • Asai K.
      • Shirai T.
      • Sato S.Y.
      • Singh R.K.
      ), and interference with TβRI or TGFβ 1 and 3 impairs breast cancer bone metastases in vivo (
      • Bandyopadhyay A.
      • Agyin J.K.
      • Wang L.
      • Tang Y.
      • Lei X.
      • Story B.M.
      • Cornell J.E.
      • Pollock B.H.
      • Mundy G.R.
      • Sun L.Z.
      ,
      • Ehata S.
      • Hanyu A.
      • Fujime M.
      • Katsuno Y.
      • Fukunaga E.
      • Goto K.
      • Ishikawa Y.
      • Nomura K.
      • Yokoo H.
      • Shimizu T.
      • Ogata E.
      • Miyazono K.
      • Shimizu K.
      • Imamura T.
      ,
      • Mourskaia A.A.
      • Dong Z.
      • Ng S.
      • Banville M.
      • Zwaagstra J.C.
      • O'Connor-McCourt M.D.
      • Siegel P.M.
      ). TβRI signals through the canonical Smad-dependent (
      • Shi Y.
      • Massagué J.
      ) or Smad-independent mechanisms (
      • Moustakas A.
      • Heldin C.H.
      ). In the Smad pathway, TβRI phosphorylates Smad2 and Smad3, which complex with Smad4 and translocate into the nucleus, acting as transcriptional modulators. TGFβ also initiates non-canonical signaling, including the mitogen-activated protein kinases (MAPKs) pathway (
      • Zhang Y.E.
      ). TGFβ-activated kinase 1 is a MAPK kinase kinase that signals through MAPK kinase (MKK) 3/6, to activate p38 and through MKK4/7 to activate JNK (
      • Yamaguchi K.
      • Shirakabe K.
      • Shibuya H.
      • Irie K.
      • Oishi I.
      • Ueno N.
      • Taniguchi T.
      • Nishida E.
      • Matsumoto K.
      ). The TGFβ-activated kinase 1/MKK6/p38 pathway was shown to be important in osteoclastogenesis (
      • Yamaguchi K.
      • Shirakabe K.
      • Shibuya H.
      • Irie K.
      • Oishi I.
      • Ueno N.
      • Taniguchi T.
      • Nishida E.
      • Matsumoto K.
      ,
      • Huang H.
      • Ryu J.
      • Ha J.
      • Chang E.J.
      • Kim H.J.
      • Kim H.M.
      • Kitamura T.
      • Lee Z.H.
      • Kim H.H.
      ,
      • Besse A.
      • Lamothe B.
      • Campos A.D.
      • Webster W.K.
      • Maddineni U.
      • Lin S.C.
      • Wu H.
      • Darnay B.G.
      ). Signaling by RANK/RANKL in osteoclasts also involves MAPKs, in particular p38 and ERK (
      • Lee Z.H.
      • Kim H.H.
      ,
      • Nakamura H.
      • Hirata A.
      • Tsuji T.
      • Yamamoto T.
      ,
      • Lee S.E.
      • Chung W.J.
      • Kwak H.B.
      • Chung C.H.
      • Kwack K.B.
      • Lee Z.H.
      • Kim H.H.
      ,
      • Gingery A.
      • Bradley E.
      • Shaw A.
      • Oursler M.J.
      ).
      We have also shown that breast cancer-derived factors sustained the activation of the osteoclastogenic transcription factor, nuclear factor of activated T cells (NFAT) c1 (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). NFAT transcription factors are controlled by the Ca2+/calmodulin-dependent phosphatase, calcineurin (
      • Rao A.
      • Luo C.
      • Hogan P.G.
      ,
      • Crabtree G.R.
      • Olson E.N.
      ). Hyperphosphorylated NFAT is restricted to the cytosol. An increase in the cytosolic free Ca2+ concentration ([Ca2+]i) activates calcineurin, which dephosphorylates NFAT, exposing the nuclear localization signal and leading to NFAT translocation to the nuclei (
      • Okamura H.
      • Aramburu J.
      • García-Rodríguez C.
      • Viola J.P.
      • Raghavan A.
      • Tahiliani M.
      • Zhang X.
      • Qin J.
      • Hogan P.G.
      • Rao A.
      ). In a majority of mature osteoclasts, treatment with RANKL results in a global elevation of [Ca2+]i (
      • Komarova S.V.
      • Pilkington M.F.
      • Weidema A.F.
      • Dixon S.J.
      • Sims S.M.
      ), whereas, in osteoclast precursors, RANKL induces Ca2+ oscillations (
      • Takayanagi H.
      • Kim S.
      • Koga T.
      • Nishina H.
      • Isshiki M.
      • Yoshida H.
      • Saiura A.
      • Isobe M.
      • Yokochi T.
      • Inoue J.
      • Wagner E.F.
      • Mak T.W.
      • Kodama T.
      • Taniguchi T.
      ). Both RANKL-induced calcium signaling and activation of NFATc1 are essential for osteoclastogenesis (
      • Takayanagi H.
      • Kim S.
      • Koga T.
      • Nishina H.
      • Isshiki M.
      • Yoshida H.
      • Saiura A.
      • Isobe M.
      • Yokochi T.
      • Inoue J.
      • Wagner E.F.
      • Mak T.W.
      • Kodama T.
      • Taniguchi T.
      ,
      • Koga T.
      • Inui M.
      • Inoue K.
      • Kim S.
      • Suematsu A.
      • Kobayashi E.
      • Iwata T.
      • Ohnishi H.
      • Matozaki T.
      • Kodama T.
      • Taniguchi T.
      • Takayanagi H.
      • Takai T.
      ,
      • Shinohara M.
      • Koga T.
      • Okamoto K.
      • Sakaguchi S.
      • Arai K.
      • Yasuda H.
      • Takai T.
      • Kodama T.
      • Morio T.
      • Geha R.S.
      • Kitamura D.
      • Kurosaki T.
      • Ellmeier W.
      • Takayanagi H.
      ,
      • Ishida N.
      • Hayashi K.
      • Hoshijima M.
      • Ogawa T.
      • Koga S.
      • Miyatake Y.
      • Kumegawa M.
      • Kimura T.
      • Takeya T.
      ). In addition to the calcineurin/NFATc1 pathway, Ca2+ is also linked to other pathways important in osteoclasts, such as protein kinase Cα (PKCα) signaling (
      • Rucci N.
      • DiGiacinto C.
      • Orrù L.
      • Millimaggi D.
      • Baron R.
      • Teti A.
      ,
      • Armstrong S.
      • Pereverzev A.
      • Dixon S.J.
      • Sims S.M.
      ,
      • Pereverzev A.
      • Komarova S.V.
      • Korcok J.
      • Armstrong S.
      • Tremblay G.B.
      • Dixon S.J.
      • Sims S.M.
      ,
      • Lee S.W.
      • Kwak H.B.
      • Chung W.J.
      • Cheong H.
      • Kim H.H.
      • Lee Z.H.
      ). Interestingly, it has been recently shown that PKCα can also activate ERK1/2 (
      • Rucci N.
      • DiGiacinto C.
      • Orrù L.
      • Millimaggi D.
      • Baron R.
      • Teti A.
      ,
      • van der Westhuizen E.T.
      • Werry T.D.
      • Sexton P.M.
      • Summers R.J.
      ,
      • Wen-Sheng W.
      ).
      In the present study, we examine the mechanisms underlying the responsiveness of osteoclast precursors to factors released by breast cancer cells. We employed mouse bone marrow cultures and RAW 264.7 murine monocytic cells for osteoclast formation, human MDA-MB-231 breast carcinoma cells, which cause bone osteolytic lesions in vivo, as a source for factors produced by the breast cancer cells, and confluent human mammary epithelial cells MCF10a as a control.

      EXPERIMENTAL PROCEDURES

      Cell Cultures

      MDA-MB-231 and MCF10a cells were kindly provided by Dr. J. Massagué (Memorial Sloan-Kettering Cancer Center, New York). MDA-MB-231 were cultured in the incubation medium (DMEM with 1.5 g/liter sodium bicarbonate, glutamine (319-020-CL, Wisent Inc.), 1 mm pyruvate (600-110-EL, Wisent Inc.), 100 units/ml penicillin, 100 μg/ml, streptomycin (450-201-EL, Wisent Inc.), 10% fetal bovine serum (080-150, Wisent Inc.)), and conditioned media (CM) was harvested after 24-h incubation. MCF10a were cultured to confluence in a 1:1 mixture of incubation medium and Ham's F-12 (Amersham Biosciences/Invitrogen) with 10% fetal calf serum, 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 0.02 μg/ml epidermal growth factor (Sigma), and CM was harvested 24 h later. MCF10a culture medium does not affect osteoclast formation (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). CM was filtered through 0.22 μm, aliquoted, and frozen at −80 °C.
      For primary osteoclast cultures, all animal studies were performed in accordance with the McGill University guidelines established by the Canadian Council on Animal Care. Mice (BALB/c, male, 6 weeks old) were purchased from Charles River Co. Bone marrow was collected from tibia and femora as described before (
      • Armstrong S.
      • Pereverzev A.
      • Dixon S.J.
      • Sims S.M.
      ) and cultured in 75-cm2 tissue culture flasks (15 × 106 cells per flask) in α-minimal essential medium with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 ng/ml human recombinant macrophage-colony stimulating factor (300-25, PeproTech Inc.). On day 1, non-adherent cells were collected, plated at 7 × 104 cells/cm2, and supplemented with macrophage-colony stimulating factor (50 ng/ml) and recombinant GST-RANKL (100 ng/ml). On day 4, fresh media with or without RANKL (100 ng/ml) or MDA-MB-231 CM (10%) was added. Macrophage-colony stimulating factor (50 ng/ml) was always present. On day 6, cells were fixed with 4% paraformaldehyde (10 min), washed with phosphate-buffered saline, and stained for tartrate-resistant acid phosphatase (TRAP) (387A-KT, Sigma).
      RAW 264.7 cells (American Type Culture Collection) were cultured in incubation medium. RAW 264.7 cells were plated at 5 × 103 cells/cm2, and 24 h later (day 1) recombinant GST-RANKL (50 ng/ml) was added. On days 2–3, cells were supplemented with fresh media with or without RANKL (50 ng/ml) or MDA-MB-231 CM (10%), cultured for 2 days, fixed, and stained for TRAP.

      Osteoclast Quantification

      Osteoclasts were identified as multinucleated (more than three nuclei) TRAP-positive cells. To evaluate the cell planar area and nuclei number of each osteoclast, images were recorded using a digital camera linked to PixeLINK Capture SE® software (PixeLINK, Ottawa, Canada).

      Test Compounds

      Cells were pretreated for 30–60 min with pharmacological or peptide inhibitors (Calbiochem): PD98059 (100 μm), Gö6976 (1 μm), GW5074 (3 μm), manumycin A (3 μm), SB203580 (1 μm), SB202474 (1 μm), SN50 (20 μm), or SN50M (20 μm). Then, the medium was replaced with fresh medium containing 10% MDA-MB-231 CM with inhibitors at the same level as during pretreatment. Using a cytotoxicity detection kit (Roche Applied Science) we have found that, at the concentrations used, the pharmacological inhibitors are not cytotoxic for osteoclast precursors. Cells were incubated with Ca2+ chelator BAPTA-acetoxymethyl ester (Invitrogen, B6769) for 10 min as described (
      • Komarova S.V.
      • Shum J.B.
      • Paige L.A.
      • Sims S.M.
      • Dixon S.J.
      ), washed, and treated with 10% MDA-MB-231 CM. Pan-specific TGFβ antibody (15 μg/ml AB-100-NA, R&D System Inc.) at a concentration identified by the supplier and previous publications (
      • Barrett J.M.
      • Rovedo M.A.
      • Tajuddin A.M.
      • Jilling T.
      • Macoska J.A.
      • MacDonald J.
      • Mangold K.A.
      • Kaul K.L.
      ), was incubated with MDA-MB-231 CM for 30 min before adding to osteoclast precursors.

      Protein Extraction, Immunoblotting, and Immunofluorescence

      Cell lysates were extracted in RIPA lysis buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 1 mg/ml aprotinin, 2 mg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, 20 mm sodium fluoride, 0.5 mm sodium orthovanadate and centrifuged at 12,000 × g for 10 min at 4 °C. Supernatant was collected, and protein was measured using a Quant-iT™ protein assay kit (Invitrogen). 20–40 μg of lysates was separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane (0.45 μm, 162-0115, Bio-Rad) using 10 mm sodium borate buffer. The membranes were blocked in 5% milk or, for p38 and p-p38, in 5% ECL advanced blocking agent (RPN418, Amersham Biosciences) in TBST buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20) for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibodies: p-Smad2 (1:1000, 3101, Cell Signaling), Smad2/3 (1:1000, 3102, Cell Signaling), Smad4 (1:100, sc-7966, Santa Cruz Biotechnology), α-tubulin (1:5000, T9026, Sigma), p-JNK (1:200, sc-6254, Santa Cruz Biotechnology), JNK (1:100, sc-81468, Santa Cruz Biotechnology), p-p38 (1:250, 9216, Cell Signaling), p38 (1:500, 9217, Cell Signaling), p-ERK1/2 (1:500, 9101, Cell Signaling), ERK1/2 (1:500, 9102, Cell Signaling), and p-PKCα/βII (1:1000, 9375, Cell Signaling). The blots were washed, incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, 170-5047; anti-rabbit, 170-5046; Bio-Rad) and visualized with a chemiluminescence system (Super signal West Pico; 34080, Pierce). For immunofluorescence, cells plated on glass coverslips were fixed with 4% paraformaldehyde and immunostaining was performed as described previously (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ) using a monoclonal antibody for p65 (1:100, sc-8008, Santa Cruz Biotechnology), biotinylated goat-anti-mouse IgG (Invitrogen) and Alexa Fluor 488-conjugated streptavidin (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen). Five random images per experimental condition were collected each containing 10–15 precursors. Cells were rated positive for nuclear localization of NF-κB if fluorescence intensity of nuclei exceeded that of the cytoplasm.

      Short Interfering RNA Transfection

      Short interfering RNA for Smad4 (5′-GGAUGGUGGACUAUGAAAUAUCTC- 3′ and 5′-GAGUAUAUUUCAUAGUCCACCAUCCUG-3′), HPRT RNA interference duplex (5′-GCCAGACUUUGUUGGAUUUGAAATT-3′ and 5′-UUCGGUCUGAAACAACCUAAACUU UAA-3′), and a double scrambled RNA interference duplex (5′-CUUCCUCUCUUUCUCUCCCUUGUG A-3′ and 5′-AGGAAGGAGAGAAAGAGAGGGAACACU-3′) were purchased from Integrated DNA Technologies (TriFECTa NM_008540). 24 h after plating RAW 264.7 cells, the medium was changed to medium without antibiotics, and 1 h later the RANKL was added to the cultures and first transfection was performed using Lipofectamine 2000 (11668-019, Invitrogen) with 10 nm of duplex. Second transfection was performed 24 h later. Protein lysates were taken at day 2 to confirm efficient Smad4 knockdown.

      Microspectrofluorometry

      Raw 264.7 cells were plated onto glass-bottom 35-mm dishes (MatTek Corp.). RANKL-primed cells were washed twice with DMEM with 10 mm HEPES and loaded at room temperature for 40 min with fura-2-AM (F1221, Invitrogen). Cells were washed twice and imaged using a fluorescence inverted microscope (T2000, Nikon), cooled charge-coupled device camera (Hamamatsu) connected to the image analysis software (Volocity, Improvision), which recorded fluorescence emission at 510 nm, following excitation at 340 and 380 nm alternated by a high speed wavelength switching device (Lambda DG-4, Quorum Technologies). [Ca2+]i values were calculated using a fura-2-AM calcium imaging calibration kit (F-6774, Invitrogen). Test compounds were added to the bath. Changes in [Ca2+]i were measured at baseline and at 30 and 60 min post stimulation.

      Statistics

      Data are presented as means ± S.E. of the mean for RAW 264.7 cells and as means ± standard deviation for bone-marrow derived osteoclasts with sample size (n) indicating the number of independent experiments for RAW 264.7 cells, or the number of osteoclasts analyzed for bone marrow-derived osteoclast. Differences were assessed by Student's t test and accepted as statistically significant at p < 0.05.

      RESULTS

      Breast Cancer-derived Factors Promote Osteoclast Fusion and Growth from RANKL-Primed Precursors

      Osteoclastogenesis was studied using primary mouse bone marrow cells and RAW 264.7 cells. When mouse bone marrow cells were treated with RANKL (100 ng/ml) for the first 3 days only (RANKL-primed), and then cultured untreated for additional 2 days, only a few small osteoclasts were observed (Fig. 1A). In contrast, supplementation of RANKL-primed precursors with RANKL (positive control, Fig. 1B) or MDA-MB-231 CM (Fig. 1C) induced marked osteoclastogenesis. In RAW 264.7 cultures, osteoclastogenesis occurs faster (within 3 or 4 days) and requires only treatment with RANKL (50 ng/ml) (
      • Akchurin T.
      • Aissiou T.
      • Kemeny N.
      • Prosk E.
      • Nigam N.
      • Komarova S.V.
      ). Similar to primary cultures, when RAW 264.7 cells were treated with RANKL for the first 1 or 2 days only (RANKL-primed), and then cultured untreated for additional 2 days, osteoclastogenesis was incomplete (Fig. 1D). Supplementation of RANKL-primed RAW 264.7 cultures with RANKL (Fig. 1E) or MDA-MB-231 CM (Fig. 1F) induced formation of many large multinucleated osteoclasts. In primary (Fig. 1G) and RAW 264.7 (Fig. 1H) cultures treatment of RANKL-primed precursors with MDA-MB-231 CM resulted in significant, 2.6- to 6.5-fold increases in osteoclast number compared with negative control. Medium conditioned by confluent normal breast cells MCF10a did not affect osteoclast formation from RANKL-primed RAW 264.7 cells (Fig. 1H). In addition, in MDA-MB-231 CM-treated cultures, osteoclast size (estimated as cell planar area) was increased 4- to 5-fold (Fig. 1, I and J), and the number of nuclei per osteoclast was increased 3-fold (Fig. 1K) compared with negative control. In MDA-MB-231 CM-treated RAW 264.7 osteoclasts, but not primary osteoclasts, the ratio of cell area/nucleus increased 2.7-fold compared with negative control (Fig. 1L). Thus, cancer-derived factors induced osteoclastogenesis from RANKL-primed precursors of bone marrow or RAW 264.7 origin.
      Figure thumbnail gr1
      FIGURE 1Breast cancer-derived factors stimulate osteoclast fusion and growth. Mouse bone marrow cells (open bars) and RAW 264.7 cells (black bars) were primed with RANKL as described in “Experimental Procedures,” and then cultured for 2 days untreated (negative control, NC), in the presence of RANKL (50 ng/ml, positive control, PC) or in the presence of MDA-MB-231 CM (10%, CM). The parallel samples were fixed and stained for TRAP. A–F, representative images of osteoclasts formed from primary bone marrow cells (A–C) and RAW 264.7 cells (D–F) in the following conditions. A and D, NC cultures; B and E, PC cultures; and C and F, MDA-MB-231-treated cultures. Scale bar of 50 μm applies to all images. G and H, treatment of primary (G) and RAW 264.7 (H) cells with MDA-MB-231 CM (CM), but not normal breast cells MCF10a CM (10a), induced a significant increase in the average number of osteoclasts compared with NC. Data are means ± S.E., n = 2–7 independent experiments, p < 0.05, as assessed by Student's t test. I–L, treatment with MDA-MB-231 CM resulted in significant increase in average size of osteoclasts (I, primary osteoclasts; J, RAW 264.7 osteoclasts), average number of nuclei per osteoclast (K), and relative cell area per single nucleus (L). For I, a cell size for n = 15–27 osteoclasts is plotted, with the horizontal bar indicating the mean of the sample. For J–L, data are means ± S.D., n = 15–27 osteoclasts for primary bone marrow cells, and means ± S.E., n = 4 independent experiments for RAW 264.7 cells, p < 0.05 (*) or 0.001 (**) as assessed by Student's t test.

      Osteoclastogenesis Induced by Breast Cancer-derived Factors Does Not Depend on the Activation of the Smad Signaling Pathway

      TGFβ acts as a permissive factor for MDA-MB-231 CM-induced osteoclastogenesis (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). We explored if the Smad pathway is involved in MDA-MB-231 CM-induced signaling in osteoclast precursors. RAW 264.7 cells were primed with RANKL (50 ng/ml), and cell lysates were collected before and 30 min after the medium was replaced with fresh medium without further additions (negative control), RANKL (positive control), or 10% MDA-MB-231 CM. No difference was observed in the levels of total Smad2/3 or phosphorylated Smad2 (supplemental Fig. S1A). Next, we silenced Smad signaling using short interfering RNA against the common mediator Smad4 (supplemental Fig. S1B). Smad4 short interfering RNA did not inhibit MDA-MB-231 CM-induced osteoclastogenesis (supplemental Fig. S1C), suggesting that the Smad pathway does not mediate this effect.

      Breast Cancer-derived Factors Induce Osteoclastogenesis in RAW 264.7 Cells by Activating ERK and p38

      We next assessed the role of ERK, JNK, and p38 in MDA-MB-231 CM-induced osteoclastogenesis. RAW 264.7 cells were primed with RANKL (50 ng/ml), then the medium was replaced with fresh medium without further additions (negative control), RANKL (50 ng/ml, positive control), MDA-MB-231 CM (10%), or MDA-MB-231 CM pretreated with pan-specific anti-TGFβ antibodies (15 μg/ml). Cell lysates were collected after 30- and 60-min incubation and immunoblotted against p-JNK, p-p38, or p-ERK1/2 (Fig. 2). Total JNK, p38, ERK1/2, and α-tubulin were used as internal and loading controls. We have found that JNK phosphorylation or total levels were not affected by any of the treatments. In contrast, MDA-MB-231 CM induced p38 phosphorylation at 30 min, which was attenuated by neutralizing TGFβ. The total level of p38 was not affected. Treatment with MDA-MB-231 CM induced ERK1/2 phosphorylation as early as 7.5 min after stimulation (data not shown). The profound ERK1/2 phosphorylation reached maximum after 15 min (data not shown) and was sustained 30 and 60 min after the addition of MDA-MB-231 CM (Fig. 2). Neutralizing TGFβ did not affect p-ERK1/2 levels 30 min after stimulation but attenuated ERK1/2 phosphorylation at 60 min (Fig. 2). Total levels of ERK1/2 were not affected by the treatments. Thus, activation of p38 and ERK1/2, but not JNK, was induced by breast cancer-derived factors in a partially TGFβ-dependent manner.
      Figure thumbnail gr2
      FIGURE 2Breast cancer derived-factors increase phosphorylation of p38 and ERK1/2 but not JNK. Cell lysates were collected from RANKL-primed RAW 264.7 cells, which were washed and incubated for 30 or 60 min in the presence of untreated culture medium (NC), RANKL (50 ng/ml, PC), MDA-MB-231 CM (10%, CM), or MDA-MB-231 CM pre-treated with the neutralizing antibodies to TGFβ (aTβ). The levels of p-JNK, p-p38, and p-ERK1/2 together with control for protein loading, α-tubulin, total JNK, p38, and ERK1/2 were assessed by immunoblotting. Shown are representative blots from one of five independent experiments.
      To explore if ERK1/2 and p38 mediate MDA-MB-231-induced osteoclastogenesis, we used pharmacological inhibitors against MEK1/2, PD98059, and against p38, SB203580. RAW 264.7 cells were primed with RANKL (50 ng/ml), then the medium was replaced with fresh medium without further additions (negative control), with RANKL (50 ng/ml, positive control), MDA-MB-231 CM (10%) alone or combined with PD98059 (100 μm), SB203580 (1 μm), PD98059 and SB203580 together, or an inactive analog of the p38 inhibitor SB202474 (1 μm). The cells were cultured for additional 2 days, fixed, stained for TRAP, and the number, size, and nucleation of osteoclasts were assessed (Fig. 3). Whereas PD98059 alone did not affect osteoclast number (Fig. 3, A and P), the p38 inhibitor SB203580 significantly reduced the number of osteoclasts formed in the presence of MDA-MB-231 CM (Fig. 3, A and S). Combination of the two inhibitors further decreased osteoclast number compared with the treatment with a single inhibitor (Fig. 3A, P+S). Even though the number of osteoclasts induced by MDA-MB-231 CM was not affected by PD98059, it induced a significant 60% reduction in osteoclast size. Inhibition of p38 reduced osteoclast size by 90%, with a decrease by 95% in the presence of a combination of ERK and p38 inhibitors (Fig. 3B). Both inhibitors, alone or in combination, reduced the osteoclast nucleation by 70–95% (Fig. 3C). Interestingly, the ratio of cell area per nucleus was increased in the presence of MEK1/2 inhibitor (Fig. 3D) but reduced in the presence of p38 inhibitor. The inactive analogue of p38 inhibitor, SB202474, did not affect osteoclast number, size, or nucleation (Fig. 3, A–D). Thus, p38 and ERK1/2 mediate cancer-induced osteoclast fusion and growth.
      Figure thumbnail gr3
      FIGURE 3Osteoclastogenic effect of breast cancer-derived factors is diminished in the presence of MAPK inhibitors. RANKL-primed RAW 264.7 cells were cultured for 2 days untreated (NC), in the presence of RANKL (50 ng/ml, PC) or in the presence of MDA-MB-231 CM (10%, CM) combined with vehicle or the MEK1/2 inhibitor, PD98059 (100 μm, P), p38 inhibitor, SB203580 (1 μm, S), the combination of these inhibitors (P+S), or an inactive analog of p38 inhibitor SB202474 (1 μm, C). The parallel samples were fixed and stained for TRAP, and the average numbers of osteoclasts (A), average size (B), average number of nuclei per osteoclast (C), and average ratio of cell area per nucleus (D) were analyzed. Data are means ± S.E., n = 3 independent experiments, p < 0.05 (*) or 0.001 (**), as assessed by Student's t test.
      To investigate a potential role of NFκB in breast cancer-induced osteoclastogenesis, we used NFκB peptide inhibitor SN50. SN50 blocked RANKL-induced nuclear translocation of p65 and RANKL-induced osteoclastogenesis (supplemental Fig. S2). In contrast, SN50 was ineffective in inhibiting MDA-MB-231 CM-induced osteoclast formation (supplemental Fig. S2C), indicating that the NFκB pathway likely affects early stages of osteoclastogenesis, which become non-critical in late osteoclast precursors.

      Breast Cancer Factor-induced Phosphorylation of ERK1/2 Is MEK1/2-dependent but Ras/Raf-independent

      Because ERK1/2 phosphorylation was only partially dependent on TGFβ, it is likely to represent a point where the synergy occurs between TGFβ and other factors produced by breast cancer cells. To analyze the signaling events leading to ERK activation, we first considered the classic Ras/Raf/MEK1/2/ERK1/2 pathway. RAW 264.7 cells were primed with RANKL (50 ng/ml) and treated with MDA-MB-231 CM alone or in combination with MEK1/2 inhibitor PD98059 (100 μm), Ras inhibitor manumycin A (3 μm), or Raf-1 inhibitor GW5074 (3 μm) for 30–60 min before cell lysates were collected and immunoblotted against p-ERK1/2. As a control, RANKL-primed cells were treated with vehicle (DMSO) or MEK1/2 inhibitor (PD98059) alone. Treatment with PD98059 abolished ERK1/2 phosphorylation both at 30 and 60 min after exposure to MDA-MB-231 CM (Fig. 4A). However, manumycin A and GW5074 did not inhibit MDA-MB-231 CM-induced ERK1/2 phosphorylation (Fig. 4B), suggesting that breast cancer factors act through a Ras/Raf-independent pathway. Both Ras and Raf1 kinase inhibitors were effective in decreasing phosphorylation of ERK1/2 in response to RANKL (Fig. 4C).
      Figure thumbnail gr4
      FIGURE 4Breast cancer factors-induced phosphorylation of ERK1/2 is blocked by MEK1/2 inhibitor, but not RAS or RAF inhibitors. Cell lysates were collected from RANKL-primed RAW 264.7 cells that were washed and incubated for 15, 30, or 60 min in the presence of vehicle (V, DMSO, 0.1%), the MEK1/2 inhibitor, PD98059 (PD, 100 μm), or MDA-MB-231 CM (10%, CM) with or without PD98059, Ras inhibitor manumycin A (MA, 3 μm) or Raf1 kinase inhibitor I GW5074 (GW, 3 μm). Phospho-ERK1/2 and total ERK1/2 together with α-tubulin, were assessed by immunoblotting. A, PD98059 inhibited the MDA-MB-231 CM-induced phosphorylation of ERK1/2. B, neither the manumycin A nor GW5074 inhibited CM-induced p-ERK1/2. C, both Ras and Raf1 kinase inhibitors decreased RANKL-induced phosphorylation of ERK1/2. Shown are representative blots from one of four independent experiments.

      Breast Cancer-derived Factors Employ PKCα to Activate ERK and Induce Osteoclastogenesis

      It has been recently shown that ERK1/2 can also be activated by PKCα (
      • Rucci N.
      • DiGiacinto C.
      • Orrù L.
      • Millimaggi D.
      • Baron R.
      • Teti A.
      ,
      • Dehvari N.
      • Isacsson O.
      • Winblad B.
      • Cedazo-Minguez A.
      • Cowburn R.F.
      ,
      • Lee M.K.
      • Pardoux C.
      • Hall M.C.
      • Lee P.S.
      • Warburton D.
      • Qing J.
      • Smith S.M.
      • Derynck R.
      ). To examine if PKCα is involved in MDA-MB-231 CM-induced ERK1/2 phosphorylation, RAW 264.7 cells were primed with RANKL (50 ng/ml), and treated with MDA-MB-231 CM alone or in combination with vehicle (DMSO), or PKCα inhibitor Gö6976 (1 μm). After 7.5, 30, and 60 min cell lysates were collected and immunoblotted against p-PKCα/βII and p-ERK1/2. MDA-MB-231 CM induced an early (7.5 min) increase in p-PKCα/βII. The increase was sustained after 30 min, and it was markedly inhibited by PKCα inhibitor Gö6976 (Fig. 5A). PKCα inhibitor also reduced ERK1/2 phosphorylation, particularly at the early time point (Fig. 5B). We next assessed if Gö6976 affects MDA-MB-231 CM-induced osteoclastogenesis (Fig. 5, C–F). Gö6976 (0.25, 0.5, and 1 μm) significantly and dose-dependently reduced the number of osteoclasts formed in the presence of MDA-MB-231 CM (Fig. 5C), profoundly decreased osteoclast size and nucleation even at the lowest concentration of an inhibitor (Fig. 5, D and E), and diminished the ratio of cell area per nucleus (Fig. 5F), suggesting that breast cancer factors act via PKCα to activate ERK and to induce osteoclast fusion and growth both in ERK-dependent and ERK-independent manner.
      Figure thumbnail gr5
      FIGURE 5Breast cancer-derived factors act via PKC to induce phosphorylation of ERK1/2 and osteoclast formation. A and B, cell lysates were collected from RANKL-primed RAW 264.7 cells that were washed and incubated for 7.5, 30, or 60 min in the presence of vehicle (DMSO, 0.1%, NC), or MDA-MB-231 CM (CM, 10%) with or without the PKC inhibitor Gö6976 (Gö, 1 μm). Phospho-PKCα/βII, p-ERK1/2, and total ERK1/2 together with α-tubulin were assessed by immunoblotting. A, treatment with MDA-MB-231 CM induced early and sustained stimulation of PKCα/βII. Gö6976 (1 μm) efficiently inhibited MDA-MB-231 CM-induced PKCα/βII phosphorylation. B, treatment with Gö6976 attenuated MDA-MB-231 CM-induced ERK1/2 phosphorylation. Shown are representative blots from one of two independent experiments. C–F, RANKL-primed RAW 264.7 cells were cultured for 2 days untreated (NC), in the presence of RANKL (50 ng/ml, PC) or in the presence of MDA-MB-231 CM (10%, CM) combined with Gö6976 at concentrations of 1, 0.5, and 0.25 μm, as indicated. The parallel samples were fixed and stained for TRAP. A significant decrease in average number of osteoclasts (C), average size of osteoclasts (D), average cell nuclei per osteoclasts (E), and average ratio of cell area per nucleus (F) was observed in samples treated with CM in the presence of 0.5–1 μm Gö6976. Data are means ± S.E., n = 3–5 independent experiments, p < 0.05 (*) or 0.001 (**) as assessed by Student's t test.

      Breast Cancer-derived Factors Induced [Ca2+]i Signaling

      Because PKCα is a Ca2+-dependent isoform, we investigated if MDA-MB-231 CM induce changes in [Ca2+]i in osteoclast precursors. RANKL-primed RAW 264.7 cells were loaded with fura-2-AM. Changes in [Ca2+]i were monitored within 2 min and 30–60 min after bath addition of DMEM, 50 ng/ml RANKL, 5–10% MDA-MB-231 CM, or 5–10% of MCF10a CM. No striking acute changes in [Ca2+]i were observed immediately upon addition of DMEM, RANKL, or CM from both cell types. However, as early as 2 min after addition of MDA-MB-231 CM, 20 ± 3% of osteoclast precursors exhibited elevations of [Ca2+]i, compared with 11 ± 3% in cultures exposed to DMEM. Although the difference did not reach statistical significance (p = 0.07), this trend was augmented at the later time points (30 and 60 min). At 30 min, significantly more cells exhibited elevations of [Ca2+]i in cultures treated with MDA-MB-231 CM and RANKL, compared with cultures treated with DMEM or MCF10a CM (Fig. 6, A and C). Neutralizing TGFβ did not reduce the number of cells exhibiting Ca2+ elevations. Cells treated with MDA-MB-231 CM or RANKL demonstrated oscillatory changes in [Ca2+]i (Fig. 6B), whereas cells treated with DMEM or MCF10a CM exhibited single elevations of [Ca2+]i. Thus, MDA-MB-231 CM, similar to RANKL (
      • Takayanagi H.
      • Kim S.
      • Koga T.
      • Nishina H.
      • Isshiki M.
      • Yoshida H.
      • Saiura A.
      • Isobe M.
      • Yokochi T.
      • Inoue J.
      • Wagner E.F.
      • Mak T.W.
      • Kodama T.
      • Taniguchi T.
      ), induced Ca2+ oscillations in osteoclast precursors.
      Figure thumbnail gr6
      FIGURE 6MDA-MB-231 CM-induced [Ca2+]i oscillations contribute to the osteoclastogenic effect of breast cancer-derived factors. A–C, RANKL-primed RAW 264.7 cells were loaded with a calcium-sensitive dye fura-2-AM. After imaging baseline [Ca2+]i, cells were incubated for a total of 60 min with untreated medium (NC), RANKL (50 ng/ml, PC), MDA-MB-231 CM (10%, CM) or MCF10a CM, and [Ca2+]i was imaged at 2, 30, and 60 min. A, representative images demonstrating [Ca2+]i in a pseudocolor with red-white corresponding to high levels of [Ca2+]i and blue-green corresponding to low levels of [Ca2+]i. B, representative traces from one of five independent experiments demonstrate changes in [Ca2+]i in three individual cells per experimental condition. C, in each experiment, the proportion of cells demonstrating changes in [Ca2+]i after 30 min of incubation in indicated conditions was analyzed. Treatment with MDA-MB-231 CM, similar to RANKL, induced significantly more oscillatory activity in osteoclast precursors compared with NC, or MCF10a-treated cells. Data are means ± S.E., n = 4–9 independent experiments, p < 0.05 (*) compared with NC and MCF10a as assessed by Student's t test. D and E, RANKL-primed RAW 264.7 cells were loaded with vehicle or calcium chelator BAPTA for 10 min, washed, and treated for 2 days with MDA-MB-231 CM (10%, CM). The parallel samples were fixed and stained for TRAP. D, treatment with BAPTA resulted in a significant decrease in the average number of osteoclasts formed in response to MDA-MB-231 CM. Data are means ± S.E., n = 6 independent experiments, p < 0.05 (*) as assessed by Student's t test. E, treatment with BAPTA resulted in a significant decrease in an average size of osteoclasts formed in response to MDA-MB-231 CM. Data are means ± S.E., expressed relative to NC, n = 4 independent experiments, p < 0.05 (*) as assessed by Student's t test.
      To further test the importance of [Ca2+]i signaling for MDA-MB-231 CM-induced osteoclastogenesis, we used a Ca2+ chelator BAPTA. We first confirmed that treatment with BAPTA prevented changes in [Ca2+]i for at least 1 h (data not shown). RAW 264.7 cells were primed with RANKL, treated with either vehicle or BAPTA for 10 min, washed, and treated with MDA-MB-231 CM (10%) for 2 days. Impediment of calcium signaling by BAPTA resulted in a significant decrease in osteoclast number and size (Fig. 6, D and E), suggesting that Ca2+ signaling is critical for MDA-MB-231-induced osteoclastogenesis.

      DISCUSSION

      We have shown that breast cancer-derived factors employ MAPKs and calcium/PKC pathways to stimulate formation of osteoclasts from RANKL-primed precursors. We have previously exposed TGFβ as a permissive but indispensable factor for these effects, and now we demonstrate that TGFβ-induced signaling does not involve the Smad pathway but acts instead through activation of MAPKs, p38 and, partially, ERK1/2. We have found that inhibition of TGFβ shortened the duration of ERK1/2 activation but did not decrease its amplitude. In contrast, inhibition of calcium-dependent PKCα resulted in suppression of cancer factor-induced phosphorylation of ERK1/2. We have found that breast cancer-derived factors, similar to RANKL, induced sustained oscillations in [Ca2+]i in osteoclast precursors. Thus, we conclude that breast cancer-derived factors induce osteoclast formation by joined activation of calcium/PKCα and TGFβ/MAPK pathways.
      Calcium signaling has been shown before to be a critical intermediary for RANKL-induced osteoclastogenic signaling (
      • Takayanagi H.
      • Kim S.
      • Koga T.
      • Nishina H.
      • Isshiki M.
      • Yoshida H.
      • Saiura A.
      • Isobe M.
      • Yokochi T.
      • Inoue J.
      • Wagner E.F.
      • Mak T.W.
      • Kodama T.
      • Taniguchi T.
      ). Interestingly, in osteoclast precursors RANKL was shown to induce delayed calcium oscillations of comparatively low amplitude (
      • Takayanagi H.
      • Kim S.
      • Koga T.
      • Nishina H.
      • Isshiki M.
      • Yoshida H.
      • Saiura A.
      • Isobe M.
      • Yokochi T.
      • Inoue J.
      • Wagner E.F.
      • Mak T.W.
      • Kodama T.
      • Taniguchi T.
      ), whereas in mature osteoclasts, RANKL induces acute global elevation of [Ca2+]i (
      • Komarova S.V.
      • Shum J.B.
      • Paige L.A.
      • Sims S.M.
      • Dixon S.J.
      ). In keeping with previous reports, we have found that priming with RANKL induced oscillations in ∼40% of RAW264.7 cells. However, when RANKL was withdrawn from the media, the number of oscillating cells declined to ∼10%. Breast cancer-derived factors were capable of supporting the [Ca2+]i oscillations in osteoclast precursors even in the absence of RANKL. This effect of breast cancer factors on calcium signaling mirrors its effect on NFATc1 activation, which we described in the previous study (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ). In addition to the calcium/calcineurin/NFATc1 pathway, we have now demonstrated that breast cancer-derived factors also stimulate calcium-dependent PKC signaling, likely acting through PKCα. We demonstrate that PKC-dependent signals cooperate with TGFβ-induced signals to stimulate ERK1/2 phosphorylation. We have found that PKCα stimulates ERK1/2 phosphorylation in a Ras and Raf-independent manner. Although not common, this pathway of ERK activation has been previously described in osteoclasts and other cell types (
      • Rucci N.
      • DiGiacinto C.
      • Orrù L.
      • Millimaggi D.
      • Baron R.
      • Teti A.
      ,
      • Wen-Sheng W.
      ,
      • Dehvari N.
      • Isacsson O.
      • Winblad B.
      • Cedazo-Minguez A.
      • Cowburn R.F.
      ). On the other hand, we have found that TGFβ signaling acts to prolong ERK activation for >1 h, so that together actions of PKC and TGFβ result in a substantial and sustained activation of ERK. Although TGFβ has been shown before to activate ERK through recruitment of adapter proteins Shc2 and Gab that activate Ras and the subsequent MAPK cascade (
      • Lee M.K.
      • Pardoux C.
      • Hall M.C.
      • Lee P.S.
      • Warburton D.
      • Qing J.
      • Smith S.M.
      • Derynck R.
      ,
      • Galliher-Beckley A.J.
      • Schiemann W.P.
      ,
      • Nishihara M.
      • Ogura H.
      • Ueda N.
      • Tsuruoka M.
      • Kitabayashi C.
      • Tsuji F.
      • Aono H.
      • Ishihara K.
      • Huseby E.
      • Betz U.A.
      • Murakami M.
      • Hirano T.
      ), in our experiments, ERK1/2 phosphorylation occurred in a Ras/Raf-independent manner, suggesting alternative mechanism for TGFβ action. One of the possibilities is that TGFβ may act to sustain stimulation of PKC, as was shown for the pancreatic cancer cells (
      • Chow J.Y.
      • Dong H.
      • Quach K.T.
      • Van Nguyen P.N.
      • Chen K.
      • Carethers J.M.
      ). Alternatively, TGFβ can signal to MAPK through TGFβ-activated kinase 1, which is linked to JNK and p38 branches of MAPK. These pathways may also cross-talk to sustain ERK activation. We have found that p38, but not JNK, was activated in osteoclast precursors by breast cancer-derived factors. Thus, we have shown that several branches of signaling, calcium/calcineurin/NFATc1, calcium/PKCα, TGFβ/p38, and PKCα+TGFβ/ERK1/2 (Fig. 7), allow osteoclast precursors to respond to breast cancer-derived factors.
      Figure thumbnail gr7
      FIGURE 7Proposed signaling pathways underlying the stimulatory effects of breast cancer-derived factors on osteoclast precursors. The asterisk refers to data obtained in the previous study (
      • Guo Y.
      • Tiedemann K.
      • Khalil J.A.
      • Russo C.
      • Siegel P.M.
      • Komarova S.V.
      ).
      We have investigated how these signaling pathways are involved in the process of breast cancer-induced formation of large multinucleated osteoclasts. Osteoclast differentiation is associated with increased expression of proteins directly needed for osteoclastic resorptive activity, such as proteolytic enzymes cathepsin K and matrix metalloproteinase 9 (
      • Boyle W.J.
      • Simonet W.S.
      • Lacey D.L.
      ). We have previously shown that osteoclasts formed in the presence of breast cancer-derived factors express appropriate levels of cathepsin K and matrix metalloproteinase 9 and are capable of bone resorption. In addition, several steps aiming at producing a giant polykaryon can be distinguished during osteoclastogenesis. First, monocytic precursors fuse to form multinucleated cells. Second, the volume of the cytoplasm increases, both due to cytoplasm gain through fusion and due to post-fusion increase in osteoclast membrane and cytoplasm volume. And third, multinucleated osteoclasts can fuse with other osteoclasts to form even bigger cells. Osteoclast size significantly affects the effectiveness of resorption (
      • Ishii M.
      • Saeki Y.
      ). Mice lacking DC-STAMP, the protein critical for osteoclast fusion, have small mononuclear osteoclasts, which are capable of bone resorption, but at a greatly reduced activity (
      • Kukita T.
      • Wada N.
      • Kukita A.
      • Kakimoto T.
      • Sandra F.
      • Toh K.
      • Nagata K.
      • Iijima T.
      • Horiuchi M.
      • Matsusaki H.
      • Hieshima K.
      • Yoshie O.
      • Nomiyama H.
      ,
      • Yagi M.
      • Miyamoto T.
      • Sawatani Y.
      • Iwamoto K.
      • Hosogane N.
      • Fujita N.
      • Morita K.
      • Ninomiya K.
      • Suzuki T.
      • Miyamoto K.
      • Oike Y.
      • Takeya M.
      • Toyama Y.
      • Suda T.
      ,
      • Vignery A.
      ). Moreover, bigger osteoclasts exhibit increased resorptive activity (
      • Lees R.L.
      • Sabharwal V.K.
      • Heersche J.N.
      ,
      • Manolson M.F.
      • Yu H.
      • Chen W.
      • Yao Y.
      • Li K.
      • Lees R.L.
      • Heersche J.N.
      ,
      • Trebec D.P.
      • Chandra D.
      • Gramoun A.
      • Li K.
      • Heersche J.N.
      • Manolson M.F.
      ) and are associated with pathological bone destruction (
      • Kaye M.
      • Zucker S.W.
      • Leclerc Y.G.
      • Prichard S.
      • Hodsman A.B.
      • Barré P.E.
      ,
      • Makris G.P.
      • Saffar J.L.
      ).
      We have found that p38, ERK1/2, and PKCα pathways contribute to different steps of osteoclast formation in a distinct manner. Inhibition of p38 led to a 2-fold decrease in osteoclast number, ∼7-fold decrease in osteoclast size and the number of nuclei per osteoclast, and 2-fold decrease in the cell area/nucleus that reflects the post-fusion osteoclast growth, demonstrating that p38 is important for monocyte fusion, osteoclast growth, and osteoclast fusion. In contrast, inhibition of ERK did not affect osteoclast number, suggesting that ERK signaling is not involved in monocyte fusion. However, ERK inhibition significantly decreased osteoclast size and nucleation while significantly increasing the cell area/nucleus. These data suggest that the ERK pathway plays an inhibitory role in the regulation of osteoclast growth while positively regulating the fusion of osteoclasts. Interestingly, the combination of inhibitors for ERK and p38 resulted in an additional decrease in osteoclast number. It has been shown previously that inhibition of ERK can induce compensatory stimulation of p38 (
      • Hotokezaka H.
      • Sakai E.
      • Kanaoka K.
      • Saito K.
      • Matsuo K.
      • Kitaura H.
      • Yoshida N.
      • Nakayama K.
      ), which can offset its potential inhibitory effects. However, we could not detect a noticeable increase in p-p38 in samples treated with MEK1/2 inhibitor (data not shown), likely suggesting that even subtle changes in balance between p38 and ERK may have significant downstream effects. We have found that chelation of Ca2+ as well as inhibition of PKCα significantly decreased osteoclast number and size. Although ERK phosphorylation was drastically reduced in the presence of PKCα inhibitor, the effects of ERK and PKCα inhibition are different, suggesting that some of osteoclastogenic effects of PKC are induced in an ERK-independent manner. For all the inhibitors, the effect on osteoclast size and nucleation was more dramatic than the effect on osteoclast number, suggesting that the initial fusion of monocytes is regulated prior to, or differently from expansion of osteoclast size either by fusion of osteoclasts or by cell growth.
      Thus, our data reveal the mechanism underlying the direct stimulatory effect of breast cancer cells on osteoclast formation. This process is complementary to other known effects of breast cancer cells on different cell types present in the bone microenvironment, such as cancer-induced stimulation of RANKL and other paracrine mediators by osteoblasts (
      • Mundy G.R.
      ,
      • Mundy G.R.
      ,
      • Guise T.A.
      ,
      • Siclari V.A.
      • Guise T.A.
      • Chirgwin J.M.
      ). Breast cancer metastases are known to preferentially establish in the skeletal sites undergoing active bone turnover (
      • Krishnamurthy G.T.
      • Tubis M.
      • Hiss J.
      • Blahd W.H.
      ,
      • Kalikin L.M.
      • Schneider A.
      • Thakur M.A.
      • Fridman Y.
      • Griffin L.B.
      • Dunn R.L.
      • Rosol T.J.
      • Shah R.B.
      • Rehemtulla A.
      • McCauley L.K.
      • Pienta K.J.
      ,
      • Schneider A.
      • Kalikin L.M.
      • Mattos A.C.
      • Keller E.T.
      • Allen M.J.
      • Pienta K.J.
      • McCauley L.K.
      ), where the numbers of osteoclast precursors primed by physiological stimuli is increased. The signaling pathways allowing osteoclast precursors to respond to the stimulation by breast cancer cells represent potential targets for the development of new therapies for osteolytic bone metastases.

      Acknowledgments

      We thank Drs. J. Massagué, Memorial Sloan-Kettering Cancer Center, and M. F. Morrison, University of Toronto, for providing reagents and cell lines used in this study. We thank Caterina Russo, Ekaterina Gusev, and Stewart Lee, McGill University, for help in obtaining preliminary data and performing preliminary data analysis.

      Supplementary Material

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