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K+-ATPase
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase Yoko Ono a, Shinsuke Chiba a, Hirohito Yano b, Noriyuki Nakayama b, Masanao Saio c, Kazuhiro Tsuruma a, Masamitsu Shimazawa a, Toru Iwama b, Hideaki Hara a, * a b c
Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, 501-1196, Japan Department of Neurosurgery, Gifu University Graduate School of Medicine, Gifu, 501-1194, Japan Department of Pathology Diagnosis, Matsunami General Hospital, Gifu, 501-6062, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 October 2016 Accepted 7 November 2016 Available online xxx
Glycoprotein nonmetastatic melanoma protein B (GPNMB), which is involved in invasion and metastasis, was found to be overexpressed in various cancers. High levels of GPNMB and Naþ/Kþ-ATPase a subunits are associated with a poor prognosis in glioblastoma patients. We showed that GPNMB interacts with Naþ/Kþ-ATPase a subunits to activate PI3K/Akt and MEK/ERK pathways. However, it remains unclear whether the interaction of GPNMB and Naþ/Kþ-ATPase a subunits is involves in progression of glioma. The tumor size induced by the injection of glioma GL261 cells was larger in transgenic mice overexpressing GPNMB when compared with wild-type mice. Additionally, the interaction of GPNMB and Naþ/Kþ-ATPase a subunits was identified in the murine glioma model and in the tumors of glioblastoma patients. Ouabain, a Naþ/Kþ-ATPase inhibitor, suppressed the glioma growth induced by the injection of glioma cells in the transgenic mice overexpressing GPNMB and blocked the GPNMB-induced migration of glioma cells. These findings indicate that GPNMB promotes glioma growth via Naþ/Kþ-ATPase a subunits. Thus, the interaction between GPNMB and Naþ, Kþ-ATPase a subunits represents a novel therapeutic target for the treatment of brain glioblastomas. © 2016 Elsevier Inc. All rights reserved.
Keywords: Glycoprotein nonmetastatic melanoma B (GPNMB) Naþ, Kþ-ATPase a subunits Glioma Glioblastoma GL261 cells
1. Introduction Glycoprotein nonmetastatic melanoma protein B (GPNMB) is also known as hematopoietic growth factor inducible neurokinin-1 type (HGFIN), dendritic cell-heparin integrin ligand (DC-HIL), and osteoactivin [1e3]. GPNMB was initially identified as a regulator of tumor growth in poorly metastatic melanoma cells [4]. GPNMB is involved in cancer cell invasion and metastasis, and it was found to be overexpressed in melanomas [5], breast cancers [6], and hepatic cancers [7]. Additionally, GPNMB mRNA and protein levels were increased in glioblastoma patients [8]. Therefore, the clarification of the mechanism may uncover a novel therapeutic approach for glioblastoma. Naþ/Kþ-ATPase is a ubiquitous transmembrane protein that maintains ion homeostasis. Naþ/Kþ-ATPase consists of a and b
* Corresponding author. Department of Molecular Pharmacology, Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigakunishi, Gifu, 501-1196, Japan. E-mail address: [email protected] (H. Hara).
subunits. The Naþ/Kþ-ATPase a subunits catalyze the iondependent ATPase activity, and its specific inhibitor is ouabain [9]. Naþ/Kþ-ATPase a subunits are increased in several cancers, including glioblastoma [10e12]. GPNMB activates the PI3K/Akt and MEK/ERK pathways via Naþ/ þ K -ATPase a subunits [13]. These pathways are important for the growth and invention of cancer cells, including glioblastoma [14]. Therefore, we hypothesized that the interaction between GPNMB and Naþ/Kþ-ATPase a subunits is important for the progression of brain glioma.
2. Materials and methods 2.1. Cell culture Murine glioblastoma GL261 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with low glucose (Nacalai Tesque, Tokyo, Japan) containing 10% fetal bovine serum (FBS; Valeant, Costa Mesa, CA, USA) in a 10-cm dish (BD Biosciences, Franklin Lakes, NJ, USA). The cells were maintained at 37 C in a 5% incubator
http://dx.doi.org/10.1016/j.bbrc.2016.11.034 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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and passaged by trypsinization every 4 days. 2.2. Murine brain glioma model Transgenic mice overexpressing GPNMB (8e12 week-old) were received an intracranial injection of 2 104 GL261 cells in 5 ml of PBS using a Hamilton microliter syringe at the following coordinates: 1 mm anterior, 2 mm lateral (left of midline) to bregma, and a depth of 3 mm from the dural surface. This protocol was completed using a stereotactic frame [24]. 2.3. Analysis of mouse brains Brains were fixed in 4% paraformaldehyde (Wako, Osaka, Japan), imbedded in paraffin (Leica Biosystems, Wetzlar, Germany), cut into 5-mm thick sections, and processed for heamatoxylin-eosin (HE) staining. Pictures were taken using an All-in-One Fluorescence Microscope (BZ-X710; Keyence, Osaka, Japan). 2.4. Immunoprecipitation assays of brain glioma mice and brain glioblastoma patients Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer's instructions. The tumor tissues of a murine glioma model and brain glioblastoma patients were homogenized in 10 ml/ g tissue ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 50 mM EDTA, 1% Triton X-100, and a protease/ phosphatase inhibitor mixture). The cell lysates were incubated
with mouse anti- Naþ/Kþ-ATPase (NKA) a1 antibody, mouse antiNKA a3 antibody, or normal mouse IgG (Santa Cruz Biotechnology). A mixture of equal parts of a protein sample and sample buffer with 20 mM DTT (Takara, Shiga, Japan) was subjected to SDSPAGE (Wako). The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P; Merck KGaA, Darmstadt, Germany). To visualize the proteins on the membrane, we used the goat anti-GPNMB antibody (1: 1000, R&D Systems Inc., Minneapolis, MN, USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000, Thermo Fisher Scientific). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan). 2.5. Human tissue specimens Tumor tissue specimens of brain glioblastoma patients were obtain from the Department of Neurosurgery, Gifu University Hospital. There were no additional preoperative interventions in the patients enrolled in the present study. The use of tumor tissues for immunoprecipitation was approved by the Institutional Review Board of Gifu University (28-14), and informed consents were acquired. The brain glioblastoma patients were men and women in their 50 se70 s. 2.6. Immunostaining of brain glioma mice Brains were fixed in 25% sucrose (Wako), imbedded in OCT
Fig. 1. Glycoprotein nonmetastatic melanoma protein B (GPNMB) overexpression promoted glioma growth in mice. (A) The sham group was only injected needle of micro syringe. (B and C) Transgenic mice overexpressing GPNMB (Tg) mice and wiled-type (WT) mice were injected 5 ml of PBS. (D and E) Tg mice and WT mice were injected with the mouse glioma GL261 cells (2 104 cells in 5 ml of PBS). Scale bar ¼ 1 mm. (F) The bar graphs represent the WT and Tg meadian tumor size 2 weeks after GL261 cells injection. Each column and bar represent the mean ± S.E.M. (n ¼ 6 or 7). *p < 0.05 vs. WT mice (Student's t-test).
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
Y. Ono et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
compound (Sakura Finetek Japan, Tokyo, Japan), cut into 10-mm thick sections and processed for immunostaining. Brain sections were incubated overnight at 4 C with the following primary antibodies, goat anti-GPNMB antibody (1:500), mouse anti-NKA a1 antibody, or mouse anti-NKA a3 antibody (1: 200). Subsequently, cells were incubated at room temperature with an Alexa-488 or Alexa-568-labeled secondary antibody (1: 1000, Thermo Fisher Scientific) for 1 h. Pictures were taken using BZ-X710 (Keyence).
2.7. Effect of Naþ, Kþ-ATPase inhibitor on glioma
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2.9. Cell migration To assess cell migration, GL261 cells were seeded in 12 well plates (BD Biosciences) with culture medium supplemented with 10% FBS. After 24 h of incubation, the culture medium was changed to DMEM containing 1% FBS. After an additional 6 h, the cells were treated with GPNMB (0.25 or 2.5 mg/ml) (R&D Systems) for 24 h. Pictures were taken at 0 and 24 h. Additionally, to determine whether GPNMB promotes cell migration via Naþ/Kþ-ATPase, we used ouabain (Enzo Life Sciences). After seeding and medium changes, the cells were treated with GPNMB (2.5 mg/ml) and ouabain for 24 h. Pictures were taken at 0 and 24 h.
The murine glioma model was injected ouabain, a Naþ/KþATPase inhibitor (Enzo Life Sciences, New York, USA) to determine whether GPNMB promotes the glioma growth via Naþ/Kþ-ATPase a subunits. Ouabain (3.6 mg/5 ml) was injected in the brain at 1day and 7 days after the injection of glioma cells.
2.10. Statistical analysis
2.8. Cell proliferation
3. Results
GL261 cells (3000 cells/well) were seeded in a 96-well plate (BD Biosciences) with culture medium supplemented with 10% FBS. After 24 h of incubation, the culture medium was changed to DMEM containing 1% FBS. After an additional 24 h, the cells were treated with of GPNMB (0.25 or 2.5 mg/ml) (R&D Systems, Minnesota, USA) for 24 h. Cell proliferation was determined using CCK-8 Assay according to the manufacturer's instructions. After 24 h of incubation, 10 ml of thawed CCK-8 solution was added to each well. Plates were incubated for 3 h at 37 C, and the absorbance was read at 450 nm with a reference wavelength of 600 nm using the microplate reader [25].
3.1. GPNMB overexpression promoted glioma growth in mice
Data are presented as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were made using a two-tailed paired student's t-test or one-way ANOVA followed by Dunnett's test, with p < 0.05 indicating statistical significance.
To identify whether GPNMB promotes glioma growth, we created a murine glioma model by injecting mouse glioma GL261 cells in the brain of transgenic mice overexpressing GPNMB (Tg) and wild-type (WT) mice. Sham and PBS injected groups did not show a change in the structure of the brain nor the formation of a tumor (Fig. 1A, B, and C). In groups injected with GL261 cells, the brain tumor size was larger 14 days after injection. Additionally, the tumor size was bigger in Tg mice compared with wild-type mice (Fig. 1D, E, and F).
Fig. 2. GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine brain glioma model. (A, D) Immunoprecipitation of GPNMB and Naþ/Kþ-ATPase a1 (NKA a1) or Naþ/Kþ-ATPase a3 (NKA a3) proteins in Tg mice and WT mice injected with murine glioma cells into the brain. (B, E) Co-immunostaining of GPNMB and Naþ/Kþ-ATPase a subunits proteins in Tg mice and WT mice injected with the murine glioma cells into the brain. Scale bar ¼ 50 mm. (C, F) Immunoprecipitation of V5-tagged GPNMB and Naþ/Kþ-ATPase a subunits proteins in Tg mice and WT mice injected the murine glioma cells into the brain.
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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Fig. 3. Ouabain, a Naþ/Kþ-ATPase inhibitor, inhibited the glioma growth. (A,B,C) Tg mice and WT mice were injected the mouse glioma GL261 cells (2 104 cells in 5 ml of PBS). Mice were treated with PBS or ouabain 1 and 7 days after the injection. Bar graphs representing the WT and Tg median tumor size 2 weeks after the injection are shown. Each column and bar represent the mean ± S.E.M. (n ¼ 3 to 7). *p < 0.05 vs. PBS treated group (Student's t-test). Scale bar ¼ 1 mm. (D) The proliferation abilities of GL261 cells treated with GPNMB (0.25 and 2.5 mg/ml). Each column and bar represent the mean ± S.E.M. (n ¼ 6). n.s.: not significant (Dunnett's t-test). (E) The migration abilities of glioma GL261 cells treated with GPNMB (0.25 and 2.5 mg/ml). Each column and bar represent the mean ± S.E.M. (n ¼ 3). *p < 0.05, **p < 0.01vs.PBS treated group (Student's t-test). Scale bar ¼ 50 mm. (F) The migration abilities of GL261 cells treated with GPNMB and ouabain. Each column and bar represent the mean ± S.E.M. (n ¼ 3). **p < 0.01 vs. vehicle group (Student's t-test).
3.2. GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine glioma model To confirm whether the interaction of GPNMB and Naþ/KþATPase a subunits effects glioma growth, we investigated immunoprecipitation and co-immunostaining in the murine glioma model. Immunoprecipitation revealed that GPNMB bound to Naþ/
Kþ-ATPase a1 and a3 in both of Tg and WT mice with glioma (Fig. 2A and D). Additionally, co-immunostaining revealed that GPNMB co-localized with Naþ/Kþ-ATPase a1 and a3 in Tg and WT mice (Fig. 2B and E). There were no differences in the Tg and WT mice for immunoprecipitation and co-immunostainig, even though there were significant differences in the degree of glioma growth between Tg and WT mice. Tg mice were injected with the V5-His-
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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Fig. 4. GPNMB interacted with Naþ/Kþ-ATPase a subunits in tumor tissues of the brain glioblastoma patients. The tumor tissues were surgically removed from brain glioblastoma patients and immunopresipitation to test whether GPNMB interacts with Naþ/Kþ-ATPase a subunits. In the tumor tissues of the brain glioblastoma patients, GPNMB bound to both Naþ/Kþ-ATPase a1 and a3 in all patients. Data from 3 patients are shown in A, B, and C.
tagged rat GPNMB cDNA construct to induced overexpression of GPNMB. We immunoprecipitated using anti-V5 antibody and detected Naþ/Kþ-ATPase using anti-Naþ/Kþ-ATPase a1and a3 antibodies. This immunoprecipitation result indicated that V5-tagged GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in Tg mice, but not WT mice. 3.3. Ouabain, a Naþ, Kþ-ATPase inhibitor, inhibited the glioma growth To clarify whether GPNMB promotes glioma growth via Naþ/KþATPase a subunits, we investigated the effects of ouabain, a Naþ/KþATPase inhibitor, against the glioma growth promoting by GPNMB. At one day and 7 days after injection of GL261 cells, mice were treated with ouabain or PBS. Ouabain inhibited the glioma growth in Tg mice injected glioma compared with the vehicle group (Fig. 3B). On the other hand, ouabain did not affect glioma growth in WT mice injected with GL261 cells (Fig. 3C). Additionally, to clarify the effects of GPNMB on cell proliferation and migration, we investigated the proliferation assay and migration assay in murine glioma GL261 cells. Although GPNMB did not affect glioma cell proliferation (Fig. 3D), it promoted the glioma cell migration in a concentration-dependent manner (Fig. 3E). Ouabain inhibited the migration stimulated by GPNMB; however ouabain alone did not affect cell migration (see Fig. 3F). 3.4. GPNMB interacted with Naþ/Kþ-ATPase a subunits in tumors from brain glioblastoma patients To clarify whether GPNMB interacts with Naþ, Kþ-ATPase a subunits in tumor tissues from glioblastoma patients, we conducted the immunoprecipitation. The tumor tissues were surgically removed from brain glioblastoma patients. GPNMB bound to both Naþ/Kþ-ATPase a1 and a3 in the tumor tissue from all of 5 patients (Fig. 4A, B, and C). We were not able to obtain the normal brain tissues from non-brain tumor patients or healthy persons to use immunopresipitation. Therefore, in this study, we were not able to investigate the immunopresipitation in normal brain tissues. 4. Discussion Here, we reported 4 major findings: 1) the brain glioma size was larger in GPNMB overexpressing mice, 2) GPNMB bound to and colocalized with Naþ/Kþ-ATPase a1 and a3 in a murine glioma model,
3) ouabain, a Naþ/Kþ-ATPase inhibitor, suppressed glioma growth, and 4) GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in tumor tissues from brain glioblastoma patients. These results are the first to demonstrate that GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine brain glioma model and brain glioblastoma patients. GPNMB is a type I transmembrane protein, and its ectodomain is shedded by ADAM10 and secreted from various cells [15]. GPNMB was increased in various cancers and promoted the growth and migration of cancer cells [6,16] and is a negative prognostic marker because it promotes cancer progression [17]. Additionally, GPNMB plays an important role in tumor progression. GPNMB mRNA and protein was upregulated in glioma or glioblastoma patients [8]. In the present study, the brain glioma size was larger in GPNMB overexpressing mice compared with WT mice (Fig. 1). Additionally, endogenous GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in both Tg and WT mice. A V5-His-tagged GPNMB construct revealed that GPNMB bound to Naþ, Kþ-ATPase a1 and a3 in Tg mice. These findings indicate that GPNMB overexpression induced glioma growth because there are no differences between Tg mice and WT mice concerning the interaction of endogenous GPNMB and Naþ/ Kþ-ATPase a subunits. Thus, overexpressed GPNMB promotes the glioma growth via Naþ/Kþ-ATPase a subunits. Naþ/Kþ-ATPase is a ubiquitous membrane protein that maintains the intracellular ion balance. Naþ/Kþ-ATPase is upregulated in various cancers [18]. Naþ/Kþ-ATPase a subunits are highly expressed in glioblastoma patients [11], indicating that Naþ/KþATPase is a potential target for therapeutic treatment of glioblastoma [19]. We reported that GPNMB interact with Naþ/Kþ-ATPase a1 and a3, and it activates PI3K/Akt and MEK/ERK pathways via Naþ/Kþ-ATPase a subunits [13]. The PI3K/Akt pathway and the MEK/ERK pathway are crucial signaling pathways for several cancers [14,20,21]. Therefore, we hypothesized that GPNMB might promote glioma growth via Naþ/Kþ-ATPase a subunits. We demonstrated that GPNMB bound to and co-localized with Naþ/KþATPase a subunits in the murine glioma model (Fig. 2). Furthermore, ouabain, as a Naþ/Kþ-ATPase inhibitor, suppressed glioma growth in GPNMB overexpressing mice, and inhibited the migration induced by recombinant GPNMB (Fig. 3). From these results, GPNMB may promote the migration and subsequent glioma growth via interactions with Naþ/Kþ-ATPase a subunits. The antitumor effects of Naþ/Kþ-ATPase inhibitors induced apoptosis and reduced migration in cancer cells [22,23]. However, in the present study, ouabain did not affect the glioma growth in WT mice injected
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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murine glioma and the migration compared with no-treated groups. These results suggest that antitumor effects against brain glioblastoma are induced by blocking the interaction between GPNMB and Naþ/Kþ-ATPase a subunits without inducing the glioma cell death. Furthermore, in the tumor tissues of brain glioblastoma patients, GPNMB bound to Naþ/Kþ-ATPase a1 and a3, suggesting that the blocking interaction between GPNMB and Naþ/ Kþ-ATPase a subunits is a promising therapeutics treatment for brain glioblastoma patients. In conclusion, our investigations indicated that GPNMB promotes glioma growth and migration via Naþ/Kþ-ATPase a subunits, suggesting that the interaction between GPNMB and Naþ/KþATPase a subunits may play a pivotal role in the poor prognosis of glioma. Therefore, the clarification of the interaction between GPNMB and Naþ/Kþ-ATPase a subunits in brain gliomas may results on a novel therapeutic target for the treatments of brain glioblastoma patients.
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Transparency document [16]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.11.034. [17]
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Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase Yoko Ono a, Shinsuke Chiba a, Hirohito Yano b, Noriyuki Nakayama b, Masanao Saio c, Kazuhiro Tsuruma a, Masamitsu Shimazawa a, Toru Iwama b, Hideaki Hara a, * a b c
Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, 501-1196, Japan Department of Neurosurgery, Gifu University Graduate School of Medicine, Gifu, 501-1194, Japan Department of Pathology Diagnosis, Matsunami General Hospital, Gifu, 501-6062, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 October 2016 Accepted 7 November 2016 Available online xxx
Glycoprotein nonmetastatic melanoma protein B (GPNMB), which is involved in invasion and metastasis, was found to be overexpressed in various cancers. High levels of GPNMB and Naþ/Kþ-ATPase a subunits are associated with a poor prognosis in glioblastoma patients. We showed that GPNMB interacts with Naþ/Kþ-ATPase a subunits to activate PI3K/Akt and MEK/ERK pathways. However, it remains unclear whether the interaction of GPNMB and Naþ/Kþ-ATPase a subunits is involves in progression of glioma. The tumor size induced by the injection of glioma GL261 cells was larger in transgenic mice overexpressing GPNMB when compared with wild-type mice. Additionally, the interaction of GPNMB and Naþ/Kþ-ATPase a subunits was identified in the murine glioma model and in the tumors of glioblastoma patients. Ouabain, a Naþ/Kþ-ATPase inhibitor, suppressed the glioma growth induced by the injection of glioma cells in the transgenic mice overexpressing GPNMB and blocked the GPNMB-induced migration of glioma cells. These findings indicate that GPNMB promotes glioma growth via Naþ/Kþ-ATPase a subunits. Thus, the interaction between GPNMB and Naþ, Kþ-ATPase a subunits represents a novel therapeutic target for the treatment of brain glioblastomas. © 2016 Elsevier Inc. All rights reserved.
Keywords: Glycoprotein nonmetastatic melanoma B (GPNMB) Naþ, Kþ-ATPase a subunits Glioma Glioblastoma GL261 cells
1. Introduction Glycoprotein nonmetastatic melanoma protein B (GPNMB) is also known as hematopoietic growth factor inducible neurokinin-1 type (HGFIN), dendritic cell-heparin integrin ligand (DC-HIL), and osteoactivin [1e3]. GPNMB was initially identified as a regulator of tumor growth in poorly metastatic melanoma cells [4]. GPNMB is involved in cancer cell invasion and metastasis, and it was found to be overexpressed in melanomas [5], breast cancers [6], and hepatic cancers [7]. Additionally, GPNMB mRNA and protein levels were increased in glioblastoma patients [8]. Therefore, the clarification of the mechanism may uncover a novel therapeutic approach for glioblastoma. Naþ/Kþ-ATPase is a ubiquitous transmembrane protein that maintains ion homeostasis. Naþ/Kþ-ATPase consists of a and b
* Corresponding author. Department of Molecular Pharmacology, Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigakunishi, Gifu, 501-1196, Japan. E-mail address: [email protected] (H. Hara).
subunits. The Naþ/Kþ-ATPase a subunits catalyze the iondependent ATPase activity, and its specific inhibitor is ouabain [9]. Naþ/Kþ-ATPase a subunits are increased in several cancers, including glioblastoma [10e12]. GPNMB activates the PI3K/Akt and MEK/ERK pathways via Naþ/ þ K -ATPase a subunits [13]. These pathways are important for the growth and invention of cancer cells, including glioblastoma [14]. Therefore, we hypothesized that the interaction between GPNMB and Naþ/Kþ-ATPase a subunits is important for the progression of brain glioma.
2. Materials and methods 2.1. Cell culture Murine glioblastoma GL261 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with low glucose (Nacalai Tesque, Tokyo, Japan) containing 10% fetal bovine serum (FBS; Valeant, Costa Mesa, CA, USA) in a 10-cm dish (BD Biosciences, Franklin Lakes, NJ, USA). The cells were maintained at 37 C in a 5% incubator
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Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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and passaged by trypsinization every 4 days. 2.2. Murine brain glioma model Transgenic mice overexpressing GPNMB (8e12 week-old) were received an intracranial injection of 2 104 GL261 cells in 5 ml of PBS using a Hamilton microliter syringe at the following coordinates: 1 mm anterior, 2 mm lateral (left of midline) to bregma, and a depth of 3 mm from the dural surface. This protocol was completed using a stereotactic frame [24]. 2.3. Analysis of mouse brains Brains were fixed in 4% paraformaldehyde (Wako, Osaka, Japan), imbedded in paraffin (Leica Biosystems, Wetzlar, Germany), cut into 5-mm thick sections, and processed for heamatoxylin-eosin (HE) staining. Pictures were taken using an All-in-One Fluorescence Microscope (BZ-X710; Keyence, Osaka, Japan). 2.4. Immunoprecipitation assays of brain glioma mice and brain glioblastoma patients Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer's instructions. The tumor tissues of a murine glioma model and brain glioblastoma patients were homogenized in 10 ml/ g tissue ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 50 mM EDTA, 1% Triton X-100, and a protease/ phosphatase inhibitor mixture). The cell lysates were incubated
with mouse anti- Naþ/Kþ-ATPase (NKA) a1 antibody, mouse antiNKA a3 antibody, or normal mouse IgG (Santa Cruz Biotechnology). A mixture of equal parts of a protein sample and sample buffer with 20 mM DTT (Takara, Shiga, Japan) was subjected to SDSPAGE (Wako). The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P; Merck KGaA, Darmstadt, Germany). To visualize the proteins on the membrane, we used the goat anti-GPNMB antibody (1: 1000, R&D Systems Inc., Minneapolis, MN, USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000, Thermo Fisher Scientific). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan). 2.5. Human tissue specimens Tumor tissue specimens of brain glioblastoma patients were obtain from the Department of Neurosurgery, Gifu University Hospital. There were no additional preoperative interventions in the patients enrolled in the present study. The use of tumor tissues for immunoprecipitation was approved by the Institutional Review Board of Gifu University (28-14), and informed consents were acquired. The brain glioblastoma patients were men and women in their 50 se70 s. 2.6. Immunostaining of brain glioma mice Brains were fixed in 25% sucrose (Wako), imbedded in OCT
Fig. 1. Glycoprotein nonmetastatic melanoma protein B (GPNMB) overexpression promoted glioma growth in mice. (A) The sham group was only injected needle of micro syringe. (B and C) Transgenic mice overexpressing GPNMB (Tg) mice and wiled-type (WT) mice were injected 5 ml of PBS. (D and E) Tg mice and WT mice were injected with the mouse glioma GL261 cells (2 104 cells in 5 ml of PBS). Scale bar ¼ 1 mm. (F) The bar graphs represent the WT and Tg meadian tumor size 2 weeks after GL261 cells injection. Each column and bar represent the mean ± S.E.M. (n ¼ 6 or 7). *p < 0.05 vs. WT mice (Student's t-test).
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
Y. Ono et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
compound (Sakura Finetek Japan, Tokyo, Japan), cut into 10-mm thick sections and processed for immunostaining. Brain sections were incubated overnight at 4 C with the following primary antibodies, goat anti-GPNMB antibody (1:500), mouse anti-NKA a1 antibody, or mouse anti-NKA a3 antibody (1: 200). Subsequently, cells were incubated at room temperature with an Alexa-488 or Alexa-568-labeled secondary antibody (1: 1000, Thermo Fisher Scientific) for 1 h. Pictures were taken using BZ-X710 (Keyence).
2.7. Effect of Naþ, Kþ-ATPase inhibitor on glioma
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2.9. Cell migration To assess cell migration, GL261 cells were seeded in 12 well plates (BD Biosciences) with culture medium supplemented with 10% FBS. After 24 h of incubation, the culture medium was changed to DMEM containing 1% FBS. After an additional 6 h, the cells were treated with GPNMB (0.25 or 2.5 mg/ml) (R&D Systems) for 24 h. Pictures were taken at 0 and 24 h. Additionally, to determine whether GPNMB promotes cell migration via Naþ/Kþ-ATPase, we used ouabain (Enzo Life Sciences). After seeding and medium changes, the cells were treated with GPNMB (2.5 mg/ml) and ouabain for 24 h. Pictures were taken at 0 and 24 h.
The murine glioma model was injected ouabain, a Naþ/KþATPase inhibitor (Enzo Life Sciences, New York, USA) to determine whether GPNMB promotes the glioma growth via Naþ/Kþ-ATPase a subunits. Ouabain (3.6 mg/5 ml) was injected in the brain at 1day and 7 days after the injection of glioma cells.
2.10. Statistical analysis
2.8. Cell proliferation
3. Results
GL261 cells (3000 cells/well) were seeded in a 96-well plate (BD Biosciences) with culture medium supplemented with 10% FBS. After 24 h of incubation, the culture medium was changed to DMEM containing 1% FBS. After an additional 24 h, the cells were treated with of GPNMB (0.25 or 2.5 mg/ml) (R&D Systems, Minnesota, USA) for 24 h. Cell proliferation was determined using CCK-8 Assay according to the manufacturer's instructions. After 24 h of incubation, 10 ml of thawed CCK-8 solution was added to each well. Plates were incubated for 3 h at 37 C, and the absorbance was read at 450 nm with a reference wavelength of 600 nm using the microplate reader [25].
3.1. GPNMB overexpression promoted glioma growth in mice
Data are presented as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were made using a two-tailed paired student's t-test or one-way ANOVA followed by Dunnett's test, with p < 0.05 indicating statistical significance.
To identify whether GPNMB promotes glioma growth, we created a murine glioma model by injecting mouse glioma GL261 cells in the brain of transgenic mice overexpressing GPNMB (Tg) and wild-type (WT) mice. Sham and PBS injected groups did not show a change in the structure of the brain nor the formation of a tumor (Fig. 1A, B, and C). In groups injected with GL261 cells, the brain tumor size was larger 14 days after injection. Additionally, the tumor size was bigger in Tg mice compared with wild-type mice (Fig. 1D, E, and F).
Fig. 2. GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine brain glioma model. (A, D) Immunoprecipitation of GPNMB and Naþ/Kþ-ATPase a1 (NKA a1) or Naþ/Kþ-ATPase a3 (NKA a3) proteins in Tg mice and WT mice injected with murine glioma cells into the brain. (B, E) Co-immunostaining of GPNMB and Naþ/Kþ-ATPase a subunits proteins in Tg mice and WT mice injected with the murine glioma cells into the brain. Scale bar ¼ 50 mm. (C, F) Immunoprecipitation of V5-tagged GPNMB and Naþ/Kþ-ATPase a subunits proteins in Tg mice and WT mice injected the murine glioma cells into the brain.
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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Fig. 3. Ouabain, a Naþ/Kþ-ATPase inhibitor, inhibited the glioma growth. (A,B,C) Tg mice and WT mice were injected the mouse glioma GL261 cells (2 104 cells in 5 ml of PBS). Mice were treated with PBS or ouabain 1 and 7 days after the injection. Bar graphs representing the WT and Tg median tumor size 2 weeks after the injection are shown. Each column and bar represent the mean ± S.E.M. (n ¼ 3 to 7). *p < 0.05 vs. PBS treated group (Student's t-test). Scale bar ¼ 1 mm. (D) The proliferation abilities of GL261 cells treated with GPNMB (0.25 and 2.5 mg/ml). Each column and bar represent the mean ± S.E.M. (n ¼ 6). n.s.: not significant (Dunnett's t-test). (E) The migration abilities of glioma GL261 cells treated with GPNMB (0.25 and 2.5 mg/ml). Each column and bar represent the mean ± S.E.M. (n ¼ 3). *p < 0.05, **p < 0.01vs.PBS treated group (Student's t-test). Scale bar ¼ 50 mm. (F) The migration abilities of GL261 cells treated with GPNMB and ouabain. Each column and bar represent the mean ± S.E.M. (n ¼ 3). **p < 0.01 vs. vehicle group (Student's t-test).
3.2. GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine glioma model To confirm whether the interaction of GPNMB and Naþ/KþATPase a subunits effects glioma growth, we investigated immunoprecipitation and co-immunostaining in the murine glioma model. Immunoprecipitation revealed that GPNMB bound to Naþ/
Kþ-ATPase a1 and a3 in both of Tg and WT mice with glioma (Fig. 2A and D). Additionally, co-immunostaining revealed that GPNMB co-localized with Naþ/Kþ-ATPase a1 and a3 in Tg and WT mice (Fig. 2B and E). There were no differences in the Tg and WT mice for immunoprecipitation and co-immunostainig, even though there were significant differences in the degree of glioma growth between Tg and WT mice. Tg mice were injected with the V5-His-
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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Fig. 4. GPNMB interacted with Naþ/Kþ-ATPase a subunits in tumor tissues of the brain glioblastoma patients. The tumor tissues were surgically removed from brain glioblastoma patients and immunopresipitation to test whether GPNMB interacts with Naþ/Kþ-ATPase a subunits. In the tumor tissues of the brain glioblastoma patients, GPNMB bound to both Naþ/Kþ-ATPase a1 and a3 in all patients. Data from 3 patients are shown in A, B, and C.
tagged rat GPNMB cDNA construct to induced overexpression of GPNMB. We immunoprecipitated using anti-V5 antibody and detected Naþ/Kþ-ATPase using anti-Naþ/Kþ-ATPase a1and a3 antibodies. This immunoprecipitation result indicated that V5-tagged GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in Tg mice, but not WT mice. 3.3. Ouabain, a Naþ, Kþ-ATPase inhibitor, inhibited the glioma growth To clarify whether GPNMB promotes glioma growth via Naþ/KþATPase a subunits, we investigated the effects of ouabain, a Naþ/KþATPase inhibitor, against the glioma growth promoting by GPNMB. At one day and 7 days after injection of GL261 cells, mice were treated with ouabain or PBS. Ouabain inhibited the glioma growth in Tg mice injected glioma compared with the vehicle group (Fig. 3B). On the other hand, ouabain did not affect glioma growth in WT mice injected with GL261 cells (Fig. 3C). Additionally, to clarify the effects of GPNMB on cell proliferation and migration, we investigated the proliferation assay and migration assay in murine glioma GL261 cells. Although GPNMB did not affect glioma cell proliferation (Fig. 3D), it promoted the glioma cell migration in a concentration-dependent manner (Fig. 3E). Ouabain inhibited the migration stimulated by GPNMB; however ouabain alone did not affect cell migration (see Fig. 3F). 3.4. GPNMB interacted with Naþ/Kþ-ATPase a subunits in tumors from brain glioblastoma patients To clarify whether GPNMB interacts with Naþ, Kþ-ATPase a subunits in tumor tissues from glioblastoma patients, we conducted the immunoprecipitation. The tumor tissues were surgically removed from brain glioblastoma patients. GPNMB bound to both Naþ/Kþ-ATPase a1 and a3 in the tumor tissue from all of 5 patients (Fig. 4A, B, and C). We were not able to obtain the normal brain tissues from non-brain tumor patients or healthy persons to use immunopresipitation. Therefore, in this study, we were not able to investigate the immunopresipitation in normal brain tissues. 4. Discussion Here, we reported 4 major findings: 1) the brain glioma size was larger in GPNMB overexpressing mice, 2) GPNMB bound to and colocalized with Naþ/Kþ-ATPase a1 and a3 in a murine glioma model,
3) ouabain, a Naþ/Kþ-ATPase inhibitor, suppressed glioma growth, and 4) GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in tumor tissues from brain glioblastoma patients. These results are the first to demonstrate that GPNMB interacted with Naþ/Kþ-ATPase a subunits in a murine brain glioma model and brain glioblastoma patients. GPNMB is a type I transmembrane protein, and its ectodomain is shedded by ADAM10 and secreted from various cells [15]. GPNMB was increased in various cancers and promoted the growth and migration of cancer cells [6,16] and is a negative prognostic marker because it promotes cancer progression [17]. Additionally, GPNMB plays an important role in tumor progression. GPNMB mRNA and protein was upregulated in glioma or glioblastoma patients [8]. In the present study, the brain glioma size was larger in GPNMB overexpressing mice compared with WT mice (Fig. 1). Additionally, endogenous GPNMB bound to Naþ/Kþ-ATPase a1 and a3 in both Tg and WT mice. A V5-His-tagged GPNMB construct revealed that GPNMB bound to Naþ, Kþ-ATPase a1 and a3 in Tg mice. These findings indicate that GPNMB overexpression induced glioma growth because there are no differences between Tg mice and WT mice concerning the interaction of endogenous GPNMB and Naþ/ Kþ-ATPase a subunits. Thus, overexpressed GPNMB promotes the glioma growth via Naþ/Kþ-ATPase a subunits. Naþ/Kþ-ATPase is a ubiquitous membrane protein that maintains the intracellular ion balance. Naþ/Kþ-ATPase is upregulated in various cancers [18]. Naþ/Kþ-ATPase a subunits are highly expressed in glioblastoma patients [11], indicating that Naþ/KþATPase is a potential target for therapeutic treatment of glioblastoma [19]. We reported that GPNMB interact with Naþ/Kþ-ATPase a1 and a3, and it activates PI3K/Akt and MEK/ERK pathways via Naþ/Kþ-ATPase a subunits [13]. The PI3K/Akt pathway and the MEK/ERK pathway are crucial signaling pathways for several cancers [14,20,21]. Therefore, we hypothesized that GPNMB might promote glioma growth via Naþ/Kþ-ATPase a subunits. We demonstrated that GPNMB bound to and co-localized with Naþ/KþATPase a subunits in the murine glioma model (Fig. 2). Furthermore, ouabain, as a Naþ/Kþ-ATPase inhibitor, suppressed glioma growth in GPNMB overexpressing mice, and inhibited the migration induced by recombinant GPNMB (Fig. 3). From these results, GPNMB may promote the migration and subsequent glioma growth via interactions with Naþ/Kþ-ATPase a subunits. The antitumor effects of Naþ/Kþ-ATPase inhibitors induced apoptosis and reduced migration in cancer cells [22,23]. However, in the present study, ouabain did not affect the glioma growth in WT mice injected
Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034
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murine glioma and the migration compared with no-treated groups. These results suggest that antitumor effects against brain glioblastoma are induced by blocking the interaction between GPNMB and Naþ/Kþ-ATPase a subunits without inducing the glioma cell death. Furthermore, in the tumor tissues of brain glioblastoma patients, GPNMB bound to Naþ/Kþ-ATPase a1 and a3, suggesting that the blocking interaction between GPNMB and Naþ/ Kþ-ATPase a subunits is a promising therapeutics treatment for brain glioblastoma patients. In conclusion, our investigations indicated that GPNMB promotes glioma growth and migration via Naþ/Kþ-ATPase a subunits, suggesting that the interaction between GPNMB and Naþ/KþATPase a subunits may play a pivotal role in the poor prognosis of glioma. Therefore, the clarification of the interaction between GPNMB and Naþ/Kþ-ATPase a subunits in brain gliomas may results on a novel therapeutic target for the treatments of brain glioblastoma patients.
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Transparency document [16]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.11.034. [17]
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Please cite this article in press as: Y. Ono, et al., Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Naþ/Kþ-ATPase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.034