Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone

  • Authors:
    • Shohei Yoshimoto
    • Hiromitsu Morita
    • Ryota Matsubara
    • Takeshi Mitsuyasu
    • Yuko Imai
    • Shunichi Kajioka
    • Masahiro Yoneda
    • Yushi Ito
    • Takao Hirofuji
    • Seiji Nakamura
    • Masato Hirata
  • View Affiliations

  • Published online on: January 19, 2016     https://doi.org/10.3892/ijo.2016.3350
  • Pages: 1258-1270
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Abstract

Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl- exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular and basal cell types. The demineralization activity of AM-1 was only 1.7% of osteoclasts demineralization activity, and the growth rate was 20% of human normal skin keratinocytes and HSC-3 cells. These results suggest that the slow expansion of several typical types of ameloblastomas in jaw bone is attributable to its slow growth and low demineralization ability.

Introduction

Ameloblastoma is a common benign odontogenic tumor worldwide and is characterized by slow but steady invasion into the maxillary and mandibular bones. A histopathological classification of ameloblastoma by the World Health Organization in 2005 defined four types: solid/multicystic, extraosseous/peripheral, desmoplastic and unicystic. Solid/multicystic ameloblastoma is further divided into follicular and plexiform types, including the basal cell type (1,2). Although ameloblastoma is not a malignant lesion, treatment of any type of ameloblastoma is limited to surgical treatments such as enucleation and resection, although recurrence with significant morbidity is common after enucleation, particularly in young people (14). Accordingly, resection remains the best way to remove ameloblastoma, although this method is not without its drawbacks. Therefore, a better understanding of the pathophysiology of ameloblastoma is necessary, because there is a high demand for drugs capable of acting as selective inhibitors of ameloblastoma.

Expansion of solid/multicystic ameloblastoma in bone is thought to occur as a result of accelerated bone resorption activities by peritumoral osteoclasts. The activation of osteoclasts is triggered by the binding of receptor activator of nuclear factor kappa-B ligand (RANKL), which is released from vicinal ameloblastoma cells, to bind receptor activator of nuclear factor kappa-B (RANK) on the plasma membrane of osteoclasts, in a manner similar to that seen in bone-invasive cancers, particularly oral squamous cell carcinoma (SCC) (58). Furthermore, several matrix metalloproteinases (MMP; MMP-1, MMP-2 and MMP-9) released from ameloblastoma cells are also involved in progression of invasive lesions similar to that seen in oral invasive SCC (915). However, ameloblastoma exhibits clinical features that differ from those of oral SCC, including its bone invasion patterns, rate of spread and clinical symptoms. For example, solid/multicystic ameloblastoma and bone invasion of SCC show clear differences on X-ray transmission images (1,16,17). The border of a solid/multicystic ameloblastoma of the jaw bone is well defined, smooth and scalloped, and the stroma exhibits a characteristic soap bubble or honeycomb appearance, often accompanied by knife-edge-like dental root resorption. In contrast, the borders of other bone invasive cancers are less well defined, often showing marked bone resorption similar to that seen in severe periodontitis, and are characterized by floating teeth without root resorption (1,16,17). These differences are likely caused by the extremely slow spread of ameloblastoma relative to that of bone-invading cancer cells derived from oral tissues, breast, lung and other organs, which tend to spread more rapidly (6).

We hypothesized that the expansion mechanism(s) of ameloblastoma in jaw bone differs from those of invasive cancer cells. In the present study, we compared the expression levels and release of RANKL in ameloblastoma and invasive oral SCC cell lines, to determine its effect on osteoclast differentiation. We also examined the possibility that ameloblastoma could directly resorb bone or dentine minerals. We found that ameloblastoma cells expressed lower amounts of RANKL than oral SCC cells but resorbed bone mineral materials by activation of vacuolar-type H+-ATPase (V-ATPase) and H+/Cl exchange transporter 7 (CLC-7) on their plasma membranes.

Materials and methods

Cell culture

The human ameloblastoma cell line AM-1 was established from a plexiform-type ameloblastoma representing typical features of native cells (18,19). Cells were grown in defined keratinocyte serum-free medium (D-KSFM; Invitrogen, San Diego, CA, USA). Human normal skin keratinocytes (HaCaT), human tongue squamous carcinoma (HSC-3), human lip fibroblasts (KD; purchased from JCRB Cell bank, Osaka, Japan), and RAW264.7 mouse macrophage cells (purchased from DS Pharma Biomedical, Osaka, Japan) were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), supplemented with 10% fetal bovine serum (FBS; PAA Laboratories, Pasching, Austria). Human osteoclast (hOC) precursor cells derived from bone marrow (purchased from Lonza, Basel, Switzerland) were grown in an original culture solution based on modified Eagle's medium (MEM) containing 100 ng/ml human synthetic RANKL (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 50 ng/ml human macrophage colony stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA). Cells were reseeded for the next passage after trypsin (Invitrogen) dispersion when they reached ~80% confluency. HaCaT and HSC-3 were gifts from M. Furue (Kyushu University, Fukuoka, Japan) and H. Takeuchi (Kyushu Dental University, Kitakyushu, Japan), respectively.

Western blot analysis

Western blots were performed as previously described (20). Briefly, cells (AM-1, HaCaT, HSC-3 KD and hOCs) were homogenized in 1 ml ice-cold lysis buffer and centrifuged at 50,000 × g for 30 min at 4°C. The supernatants (20 μg) were then separated on 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinyldifluoride membranes (Millipore, Darmstadt, Germany). Immunoblot analyses were performed using mouse anti-human RANKL monoclonal antibody (1:500; Sigma-Aldrich, St. Louis, MO, USA), mouse antihuman V-ATPase isoform α3 monoclonal antibody (V-ATPase α3 or TCIRG1; 1:500; Abcam, Cambridge, MA, USA), rabbit anti-human CLC-7 (1:500; Abgent, Inc., San Diego, CA, USA), rabbit anti-human chloride transporter 3 (CLC-3 polyclonal antibody, 1:500; Abcam), or mouse anti-human cathepsin K monoclonal antibody (1:1,000; Sigma-Aldrich). Rabbit antihuman β-actin monoclonal antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA) was used as an internal standard. Blots were developed with horseradish peroxidase (HRP)-linked secondary antibodies (1:3,000; Cell Signaling Technology) and visualized using the enhanced chemiluminescence (ECL) system, LAS-4000 (GE Healthcare, Cleveland, OH, USA). Immobilon western chemiluminescent HRP substrate (Millipore) was used for detection. For the biotinylation assay, cells (AM-1 and HaCaT; 80% confluent in a 6-cm dish) were lysed in lysis buffer following incubation in cold biotin reagent (1 mg/ml sulpho-NHS-SS-biotin; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 4°C, and centrifuged at 50,000 × g for 30 min at 4°C. The supernatant was then incubated with 300 μl cold avidin beads (Thermo Fisher Scientific) at 4°C for 2 h, followed by centrifugation at 3,500 × g for 30 min at 4°C. Supernatants were then aspirated, and the beads were washed three times with 1 ml cold lysis buffer, once with cold 500 mM NaCl and Tris-HCl (pH 7.5), and once with cold 10 mM Tris-HCl (pH 7.5). The beads were boiled with 50 μl Laemmli sample buffer, and a 20-μl aliquot was analyzed by SDS-PAGE. Antibodies used for the immunoblot analysis included anti-V-ATPase α3, anti-CLC-7 and anti-E-cadherin (rabbit anti-human monoclonal; 1:1,000; Cell Signaling Technology).

Coculture and osteoclastogenesis experiments

AM-1 and KD cells were cocultured at a ratio of 1:1 in a 6-cm dish in a 1:1 mixture of D-KSFM and 10% FBS-containing α-MEM, as previously described (5,21). Bone marrow cells were collected from C57BL/6J mice at 6 weeks of age. Cells (1.5×105)/well in 24-well plates were cultured in 10% FBS-containing α-MEM with 20 ng/ml M-CSF. After 2 days, adherent cells were used as bone marrow-derived monocyte/macrophage precursor cells (BMM). AM-1 or HSC3 cells were cocultured with RAW264.7 cells or BMM in 24-well plastic plates at a ratio of 1:5 in 5% FBS-containing α-MEM medium (α-MEM), or a mixture of D-KSFM and 10% FBS-containing α-MEM (mixed medium). All media were supplemented with 50 ng/ml M-CSF. As a positive control, 100 ng/ml RANKL was added to the media. After 5–7 days, cells were fixed in 4% paraformaldehyde (PFA), and stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP kit (Sigma-Aldrich). TRAP-positive multinuclear cells containing more than three nuclei were considered to be osteoclasts (22). For assessment of osteoclast differentiation, RAW264.7 cells (5×104 cells/well) were cultured in 24-well plates in α-MEM. PP2, PP3 (Abcam), and (3Z)-3-[(1-Methylindol-3-yl)methylidene]-2-oxo-1H-indole-5-sulfonamide [spleen tyrosine kinase (Syk) inhibitor (Syk inh); Abcam] were applied 1 h prior to changing the medium to mixed medium. Recombinant mouse semaphorin 3A (R&D Systems, Minneapolis, MN, USA) was applied 12 h prior to changing the medium to the mixed medium. The culture medium was changed every second day. After 7 days, cells were fixed in 4% PFA and stained for TRAP. The method for counting TRAP-positive cells was as described above.

Cell observation and pit assay

Calcium phosphate- and collagen I-coated coverslips, and 24-well calcium phosphate-coated plates (BD BioCoat Osteologic; BD Biosciences, San Jose, CA, USA; and Osteo Assay surface; Corning Incorporated, Corning, NY, USA, respectively) were used for the pit assay (23). AM-1 and HaCaT cells were cultured on Osteologic coverslips for 2–10 days. AM-1 and hOCs were cultured on osteo assay surface plates for 10 days. Cells and pits were observed at magnifications of 4× and 10× with an inverted microscope (IX71; Olympus, Tokyo, Japan); images were captured using cellSens imaging software (ver. 1.7.1; Olympus). The pits in three samples were counted in random regions with pit areas quantified using ImageJ software (NIH, Bethesda, MD, USA).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from AM-1 using TRIzol reagent (Thermo Fisher Scientific). Isolated total RNA (4 μg) was subjected to RT-PCR analysis using PCR Super Mix High Fidelity (Thermo Fisher Scientific) and the CLC-1 primers 5′-ctgagccagcctgtctgtttt-3′ (forward) and 5′-ctccaactcgccctc tacctt-3′ (reverse); CLC-2 primers 5′-tagccctgaggcttctgtctg-3′ (forward) and 5′-ggagcaggatcaattttgcag-3′ (reverse); CLC-3 primers 5′-tagggcaaatattgcctggtg-3′ (forward) and 5′-gatggaaccttgatgccaaaa-3′ (reverse); CLC-4 primers 5′-ctcctcccatacaaagggacac-3′ (forward) and 5′-taatgctgtcctcctgtgctgt-3′ (reverse); CLC-5 primers 5′-gcatatagcacagatggcgaac-3′ (forward) and 5′-acggttggaatttctcttgcat-3′ (reverse); CLC-6 primers 5′-ctggaatgggagacagaggtg-3′ (forward) and ′5-cctccatggtccagtcttcac-3′ (reverse); CLC-7 primers 5′-gactcgtagcaccagggtttg-3′ (forward) and 5′-catgtgctaggggaagacctg-3′ (reverse); CLC-Ka primers 5′-gaggaggtggtcaaggttgtg-3′ (forward) and 5′-ttctcaggagcctctcactgg-3′ (reverse); and CLC-Kb primers 5′-gaggaggtggtcaaggttgtg-3′ (forward) and 5′-tttcttcatctccacccagga-3′ (reverse). Total RNA extracted from HEK293 (kindly provided by Dr H. Takeuchi) or human skeletal muscle (Agilent Technologies, Santa Clara, CA, USA) was used as a positive control.

Fluorescent immunohistochemistry

Clinical sample collection was performed at the Department of Oral and Maxillofacial Surgery, Kyushu University Hospital (Fukuoka, Japan). Specimens were removed surgically from three patients with primary ameloblastoma (plexiform, follicular and basal cell types); all patients provided informed consent before enrollment. Immunohistochemistry was performed as previously described (20). Briefly, following the initial biopsy, all specimens were fixed in 4% PFA in phosphate-buffered saline (pH 7.4) overnight, embedded in paraffin wax and sectioned at 5 μm. After deparaffinization and blocking procedures, specimens were stained with primary antibodies (mouse anti-human V-ATPase α3 antibody, 1:200; rabbit anti-human CLC-7 antibody, 1:200) and secondary antibodies (Alexa Fluor 594 conjugated anti-mouse IgG, HRP-linked antibody, 1:1,000; Alexa Fluor 488 conjugate anti-rabbit IgG, HRP-linked antibody, 1:1,000; Invitrogen). Sections were then mounted using PermaFluor mountant (Lab Vision Products, Thermo Fisher Scientific) and visualized at the appropriate wavelength using a fluorescence microscope (BioRevo BZ-9000; Keyence). For cell staining, AM-1 cells and hOCs were fixed in 4% PFA. After permeabilization with digitonin (100 μg/ml; Wako Pure Chemical) and blocking with 2.5% bovine serum albumin (Sigma-Aldrich), cells were stained with the aforementioned primary antibodies (mouse anti-human E-cadherin antibody, 1:200; BD Biosciences; rabbit anti-human CLC-3 antibody, 1:200; Abcam; mouse anti-human V-ATPase α3 antibody, 1:200; rabbit anti-human CLC-7 antibody, 1:200). This was followed by incubation with secondary antibodies, and then mounting with PermaFluor mountant for visualization. Single-cell samples were visualized with a confocal microscope (LSM700; Carl Zeiss, Oberkochen, Germany; or A1; Nikon, Tokyo, Japan). Images were processed using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA).

Drugs

Bafilomycin A1 was obtained from Merck. All other chemicals were purchased from Sigma-Aldrich.

Statistical analyses

All data are expressed as the mean ± standard error of the mean (SEM). Student's t-test and one-way analysis of variance (ANOVA) were used for statistical evaluations. Statistical significance was set as P<0.05.

Results

Assessment of bone demineralization and RANKL expression by AM-1 cells

Western blot analysis using anti-RANKL antibody was performed to detect expression of RANKL in AM-1, HSC-3 and HaCaT cells. HSC-3, bone invasive cancer cells and HaCaT, normal skin keratinocyte, were used as a positive and negative control, respectively. RANKL expression was high in HSC-3 cells, whereas in AM-1 cells, its expression was similar to that in the negative control (Fig. 1A). To evaluate the effect of tumor-stromal interactions on RANKL expression by ameloblastoma cells, AM-1 and KD cells (human lip fibroblasts) were cocultured in mixed medium, but this did not increase RANKL expression (Fig. 1B). Next, we examined the osteoclastic differentiation of BMM and RAW264.7 cells cocultured with AM-1 or HSC-3 cells grown in a medium of α-MEM alone or α-MEM plus D-KSFM (mixed medium); as a positive control, 100 ng/ml RANKL was added to the medium in the absence of coculture cells. TRAP-positive multinuclear cells were detected in the cocultures with HSC-3 cells, but not in those with AM-1 grown in α-MEM medium (Fig. 2). On the other hand, cocultures grown in mixed medium produced more TRAP-positive cells, which were inhibited to the control level by the addition of osteoprotegerin (OPG; 100 ng/ml) (Fig. 2C and D), indicating the presence of RANKL-independent osteoclastogenesis in the mixed medium. To investigate the RANK-independent osteoclastogenesis of RAW264.7 in mixed medium, we tested several inhibitors of non-receptor tyrosine kinases, which are related to immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor pathways. PP2, a specific Src kinase family inhibitor, inhibited the formation of TRAP-positive multinuclear cells in a dose-dependent manner (Fig. 3A). In contrast, PP3 (50 μM), a negative control for PP2, did not detectably inhibit TRAP-positive multinuclear cell formation. However, semaphorin 3A (sema3A; >1 μg/ml), a negative regulator of the formation of plexin-A1-triggering receptor expressed in myeloid cells-2 (TREM-2), which is an immunoreceptor, DNAX activating protein of 12-kDa (DAP12) complex, and Syk inh (50 μM) partially inhibited the RANKL-independent TRAP-positive cell formation (Fig. 3). Interestingly, further inhibition was observed by application of a mixture of sema3A (2 μg/ml) and Syk inh (50 μM) (Fig. 3B).

AM-1 cells dissolve the mineral substrate

Based on the above results, we hypothesized that ameloblastoma cells might directly demineralize bone. Their demineralization ability was assessed using calcium phosphate- and collagen I-coated coverslips (Osteologic), which mimic bone mineral substrate. AM-1 cells grew very slowly and more than 10 days were needed to reach ~80% confluency after an initial seeding of 6.0×105 cells in a non-coated 6-cm dish. After reaching confluency, cells (2.0×105 cells) were reseeded on Osteologic coverslips in 24-well culture plates and cultured for an additional 10 days. Small round pits with a diameter of 30–40 μm were observed on the coverslips at a density of 47±3 pits/mm2 (n=3; Fig. 4A, left panel). In contrast, HaCaT cells grew quickly, similarly to HSC-3 cells, reaching ~80% confluency within 2 days of culture in DMEM containing 10% FBS. HaCaT cells produced no pits on Osteologic coverslips, even after reaching full confluency (Fig. 4A). Addition of bafilomycin A1, an V-ATPase inhibitor, to the culture medium inhibited pit formation in a dose-dependent manner at concentrations ranging from 1 to 10 nM (Fig. 4C and D). Addition of 10 nM concanamycin, another V-ATPase inhibitor, also completely inhibited pit formation by AM-1 cells (data not shown). Next, we determined the demineralization ability of AM-1 cells compared with hOCs, which were differentiated from bone marrow-derived osteoclast precursor cells using RANKL. AM-1 and hOCs were initially seeded at a density of 1×104 cells on Osteologic coverslips. Cells were then cultured for 10 days, and the absorbed pits were analyzed as described above. The mean pit area produced by AM-1 cells was 1.7% of that produced by hOCs (0.1±0.03% for AM-1 vs. 5.9±2.5% for hOCs; n=3 for each treatment; Fig. 4B). The addition of bisphosphonates, either alendronate (10 μM) or pamidronate (10 μM), which are inhibitors of osteoclasts, had no inhibitory effect on the viability of AM-1 cells, although the RAW264.7 cells were susceptible (data not shown).

V-ATPase and CLC-7 are expressed on the plasma membrane of AM-1 cells

Based on the inhibitory effects of bafilomycin A1 and concanamycin, we assumed that demineralization of calcium phosphate by AM-1 cells could be caused by V-ATPase, a proton pump expressed on the surface of the plasma membrane. V-ATPase is coexpressed with the chloride transporter CLC-7 on the surface of organelles, such as lysosomes, in most eukaryotic cells, and on the plasma membrane of osteoclasts. Western blot analysis of AM-1 cell lysates showed the presence of V-ATPase α3 at levels similar to that of hOCs, while CLC-7 was present at levels greater than those in hOCs and HaCaT cells. The relative expression of V-ATPase and CLC-7 was hOCs ≈ AM-1 >HaCaT and AM-1 >hOCs ≈ HaCaT, respectively (Fig. 5A). To examine whether these proteins were present on the cell-surface, we labeled intact AM-1 and HaCaT cells with a biotinylation reagent, followed by lysis and detection with streptavidin. Biotinylated V-ATPase α3 and CLC-7 were observed in the cell-surface extracts of only AM-1 cells but were absent in HaCaT cells (Fig. 5B). As a positive control, E-cadherin, a transmembrane protein in the plasma membrane, was detected in the cell-surface extracts of both AM-1 and HaCaT cells (Fig. 5B). In addition, the expression profile of the CLC chloride channel family in AM-1 cells was assessed by RT-PCR analysis; the results showed that several mRNA for CLC-2, CLC-3, CLC-7 and CLC-Kb were detected (Fig. 6). Immunofluorescence analysis of CLC-3 demonstrated that the positive signals were localized almost entirely in the cytosol of AM-1 cells (Fig. 7).

Comparison of osteoclastogenic features of AM-1 cells and osteoclasts

Immunofluorescence analysis was performed to examine the membrane localization of both V-ATPase α3 and CLC-7 (Fig. 8, Z-axis and tiled images). Merged images of V-ATPase α3 and CLC-7 are shown in Fig. 8C and F. As a positive control, an identical pattern of membrane localization of V-ATPase α3 and CLC-7 was evident in hOC (Fig. 9A). Nevertheless, the cytosol of both AM-1 cells and hOCs was also stained by antibodies for V-ATPase α3 and CLC-7, because these factors are usually expressed in the lysosomes of all eukaryotic cells (Figs. 8 and 9A). Next, we performed TRAP staining of AM-1 cells. AM-1 cells were negative for TRAP, in clear contrast to the findings for osteoclasts (data not shown). Furthermore, neither AM-1 nor HaCaT cells expressed cathepsin K, a cysteine proteinase released from osteoclasts to digest the organic materials of bone (Fig. 9B).

Distribution of V-ATPase and CLC-7 in clinical specimens of ameloblastoma lesions

Finally, we performed immunofluorescence staining of clinically dissected specimens from patients bearing three types of ameloblastoma: plexiform, follicular and basal cell types. All types of solid/multicystic ameloblastoma specimens were clearly stained with V-ATPase α3 and CLC-7 antibodies in the cytosol, particularly on the plasma membrane of the epithelium rather than the stroma (Fig. 10).

Discussion

Disruptive bone resorption as a result of invasion by cancer cells is caused by maturation and functional activation of osteoclasts by RANKL released from cancer cells (7,24,25). Ameloblastoma cells are thought to behave in a similar manner, expressing RANKL on the plasma membrane, thereby activating peripheral osteoclasts (5), suggesting that the mechanisms for ameloblastoma expansion in jaw bone are similar to those of bone-invasive cancers. However, despite these apparent similarities, the clinical features of bone cancer and ameloblastoma are quite different (3,6), which led us to examine the osteoclastogenic responses induced by ameloblastoma cells. We found that the expression and release of RANKL by AM-1 cells, an ameloblastoma cell line, appeared to be too low to activate osteoclasts, even in the presence of other cell types such as fibroblasts. Many reports have highlighted the importance of the tumor-stroma interaction in tumor cell invasion, and the role of osteoclastogenesis in odontogenic tumors; however, no increases in RAW264.7 differentiation or RANKL expression in AM-1 were observed in cocultures with AM-1 or KD cells, respectively (2630). In fact, Kumamoto and Ooya (31) reported little expression of RANKL in either plexiform or follicular ameloblastoma specimens. In addition, RANKL-positive cells were shown by qualitative and quantitative analysis of immunoreactivity to be distributed more commonly throughout the stroma rather than in the epithelium surrounding the ameloblastoma (32). RANKL and OPG positive cells were also more commonly found in the stromal cells of ameloblastoma, implying that RANKL expression in ameloblastoma may play a key role in the proliferation and tumor progression of associated stromal cells, similar to that seen in breast cancer (33,34).

However, Sandra et al (5) and Kibe et al (21) demonstrated osteoclastic differentiation in cocultures of RAW264.7 macrophages with AM-3 cells, a follicular-type ameloblastoma cell line, and with AM-1 cells, results that differ from those of the present study. Differences in the culture medium used in the present study may be a possible explanation for this difference: untreated RAW264.7 cells cultured in a mixed medium (α-MEM plus D-KSFM) alone, which was used in the studies by both Sandra et al (5) and Kibe et al (21), differentiated into TRAP-positive multinuclear cells. In contrast, when cultured in α-MEM alone, BMM and RAW246.7 cells did not differentiate into TRAP-positive cells without the support of HSC-3 cells or added RANKL. Recently, it has been identified that osteoclastic differentiation comprises two different types of receptor-mediated signaling pathways, including both RANK and immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptors (35). ITAM-bearing receptors are classified into two types: Fc receptor common γ subunit (FcRγ) receptors [osteoclast-associated receptor (OSCAR) and paired immunoglobulin-like receptor-A (PIR-A)] and DAP12 receptors [signal-regulatory protein b1 (SIRPβ1) and TREM-2]. ITAM-bearing receptors commonly activate phosphorylation of Syk and Src kinases, which are members of the non-receptor tyrosine kinase family (36). The present study showed that osteoclast formation by RAW264.7 in a mixed medium was completely inhibited by PP2 (>20 mM). Src kinases are related not only to the activation of ITAM-bearing receptors, but also to activation of M-CSF receptors (37,38). M-CSF and its receptors are involved in an initial step of differentiation (38). Consequently, an Src kinase inhibitor fully suppressed osteoclastic differentiation, while sema3A (>1 μg/ml), a negative regulator of plexin-1A-TREM-2-DAP12 complex formation, and Syk inh (50 μM), an inhibitor of FcR, partially inhibited RAW264.7 differentiation (39,40). Together, these results indicate that sema3A suppresses DAP12 receptor signaling, while Syk inh suppresses FcRγ signaling. Therefore, we conclude that the D-KSFM used in the previous studies (5,21) must contain some ligands for M-CSF receptors and ITAM-bearing receptors, thus promoting osteoclastogenesis.

From these observations, we hypothesized that ameloblastoma might itself be able to resorb bone, given the fact that their radiographic images show well-defined tumor margins and knife-edge-like dental root resorption, in clear contrast to the images of bone-invasive cancers (1). We speculate that there is little space for osteoclasts to exist between the peritumor wall of ameloblastoma and the dental roots, a feature that is far more typical of ameloblastoma than that of other bone-related tumors. We demonstrated that AM-1 cells formed resorption pits even in the absence of osteoclastic cells, and at a magnitude much greater than that of osteoclasts alone, indicating the probable activation of V-ATPase and CLC-7 expressed on their plasma membranes. Thus, AM-1 exhibited bone demineralization activities similar to osteoclasts. However, AM-1 showed differing features from osteoclasts with regard to the absence of TRAP staining, the insensitivity to bisphosphonates and lack of cathepsin K expression. These results support direct bone demineralization of AM-1, but not experimental contamination of osteoclasts.

In osteoclasts, the α3 subunit of V-ATPase is thought to be essential for bone resorption (4144), because its absence results in autosomal recessive osteopetrosis (45,46). We thus examined the α3 subunit as a marker of V-ATPase on the plasma membrane of AM-1 cells and clinical specimens. V-ATPase inhibitors bafilomycin A1 and concanamycin effectively blocked pit formation, indicating that AM-1 cells have the ability to resorb calcium phosphate with V-ATPase, a mechanism similar to that of osteoclasts. However, CLC-7 is also a key regulator of bone demineralization, releasing Cl in cooperation with H+ release by V-ATPase in osteoclasts (44). We also detected CLC-7 expression on the plasma membrane of both AM-1 cells and ameloblastoma clinical specimens. Thus, we showed that ameloblastoma cells demineralized bone matrix by means of V-ATPase and CLC-7 expressed on their plasma membranes. Among CLC chloride channel family members, CLC-1, 2, Ka and Kb are classified into voltage-activated chloride channels, while CLC-3, 4, 5, 6 and 7 as chloride transporters. An isotype of these chloride transporter proteins, CLC-3, acts as a main chloride transporter in the endosomes and lysosomes of ameloblastoma cells and other cell types (47).

Taken together, these data show that ameloblastoma cells appear to resorb bone in a manner similar to that of osteoclasts, but at only 1.7% of the activity. Furthermore, AM-1 cells grew considerably slower than other cell lines, such as HaCaT and HSC-3, a phenotype likely related to their slow expansion in the jaw bone. The organic bone matrix consists of collagen, bone Gla protein, bone sialoprotein and other proteins. This complex is degraded by several MMP (MMP-1, MMP-2 and MMP-9), which are released from ameloblastoma, but the cathepsin K released from osteoclasts was not found in ameloblastoma (9,10,1214,46). Remarkably, both MMP-2 and MMP-9 are abundantly expressed in AM-1 and AM-3 cells relative to their expression in other oral cancer cells (21,48). These findings appear to suggest that ameloblastoma can resorb bone and dental roots, including the periapical tissue, in concert with their activity in resorption of inorganic hard tissues.

In conclusion, we demonstrated direct bone demineralization, possibly by activation of V-ATPase plus CLC-7 present on the plasma membrane of ameloblastoma rather than by RANKL release by ameloblastoma cells, and therefore we propose a new concept for the slow pathophysiological progression of several typical types of ameloblastomas.

Acknowledgements

The present study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI, #24592991 to H.M., #26861732 to Y.I. and #24229009 to M.H.). We would like to thank Professors K. Okabe and H. Takeuchi for critical comments and Professors T. Furue and H. Takeuchi for providing us with HaCaT and HSC-3 cells, respectively. We also thank Drs M. Matsuda, H. Umebayashi, T. Kawakubo-Yasukochi, A. Mizokami, H. Kondo and S. Kanda for the helpful comments and technical assistance. We would also like to thank the Research Support Center, Graduate School of Medical Sciences, Kyushu University for technical support in using the Carl Zeiss LSM700 and Nikon A1 laser scanning confocal microscopes.

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March 2016
Volume 48 Issue 3

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Copy and paste a formatted citation
APA
Yoshimoto, S., Morita, H., Matsubara, R., Mitsuyasu, T., Imai, Y., Kajioka, S. ... Hirata, M. (2016). Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone. International Journal of Oncology, 48, 1258-1270. https://doi.org/10.3892/ijo.2016.3350
MLA
Yoshimoto, S., Morita, H., Matsubara, R., Mitsuyasu, T., Imai, Y., Kajioka, S., Yoneda, M., Ito, Y., Hirofuji, T., Nakamura, S., Hirata, M."Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone". International Journal of Oncology 48.3 (2016): 1258-1270.
Chicago
Yoshimoto, S., Morita, H., Matsubara, R., Mitsuyasu, T., Imai, Y., Kajioka, S., Yoneda, M., Ito, Y., Hirofuji, T., Nakamura, S., Hirata, M."Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone". International Journal of Oncology 48, no. 3 (2016): 1258-1270. https://doi.org/10.3892/ijo.2016.3350