VNUT/SLC17A9, a vesicular nucleotide transporter,
regulates osteoblast differentiation
Asako Inoue1, Kayoko Nakao-Kuroishi1 , Kaori Kometani-Gunjigake1, Masahiro Mizuhara1,
Tomohiko Shirakawa1, Misa Ito-Sago1, Kazuma Yasuda1, Mitsushiro Nakatomi2, Takuma Matsubara3,
Yukiyo Tada-Shigeyama4, Kazumasa Morikawa5, Shoichiro Kokabu3 and Tatsuo Kawamoto1
1 Division of Orofacial Functions and Orthodontics, Department of Health Improvement, Kyushu Dental University, Kitakyushu-shi, Japan
2 Division of Anatomy, Department of Health Improvement, Kyushu Dental University, Kitakyushu-shi, Japan
3 Division of Molecular Signaling and Biochemistry, Department of Health Improvement, Kyushu Dental University, Kitakyushu-shi, Japan
4 Division of Dental Anesthesiology, Department of Science of Physical Functions, Kyushu Dental University, Kitakyushu-shi, Japan
5 Division of Pediatric and Special Care Dentistry, Department of Developmental Oral Health Science, School of Dentistry, Iwate Medical
University, Morioka, Japan

Keywords
ATP; compressive force; osteoblast;
osteoblast differentiation; P2 receptor;
VNUT
Correspondence
K. Nakao-Kuroishi, Division of Orofacial
Functions and Orthodontics, Department of
Health Improvement, Kyushu Dental
University, 2-6-1 Manazuru, Kokurakita-ku
Kitakyushu-shi 803-8580, Japan
Fax: ＋81 93 571 2033
Tel: ＋81 93 582 1131
E-mail: kayo-na■kyu-dent.ac.jp(Received 30 January 2020, revised 4 June
2020, accepted 22 June 2020)
doi:10.1002/2211-5463.12918

Osteoblasts release adenosine triphosphate (ATP) out of the cell following
mechanical stress. Although it is well established that extracellular ATP
affects bone metabolism via P2 receptors [such as purinergic receptor P2X7
(P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP
release from osteoblasts remains unknown. Recently, a vesicular nucleotide
transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that
preserves ATP in vesicles has been identified. The purpose of this study
was to elucidate the role of VNUT in osteoblast bone metabolism. mRNAand protein expression of VNUT were confirmed in mouse bone and inosteoblasts by quantitative real-time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3-E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased
concomitant with an increase in extracellular ATP levels. Furthermore,compressive force decreased the osteoblast differentiation capacity of
MC3T3-E1 cells. shRNA knockdown of Slc17a9 in MC3T3-E1 cells
reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR
analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations,knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading.
In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response tocompressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity.

Bone homeostasis is maintained by the balanced activityof bone-forming osteoblasts and bone-resorbing osteoclasts. Mechanical stress and loading are key factors forthe regulation of bone homeostasis [1]. Osteoblasts cansense mechanical stress and respond either directly or
via coordinated crosstalk with osteoclasts [2–6]. Long

Abbreviations
ALP, alkaline phosphatase; ATP, adenosine triphosphate; P2X1R, purinergic receptor P2X1; P2X7R, purinergic receptor P2X7; P2Y2R,
purinergic receptor P2Y2; qPCR, quantitative real-time PCR; SLC17A9, solute carrier family 17 member 9; VNUT, vesicular nucleotide
transporter.

1612

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

A. Inoue et al.

periods of inadequate mechanical loading, for example,
during continued bed rest or the microgravitational
environment of space flight, result in bone loss [7,8]. In
orthodontic treatment, asymmetric mechanical loading
to alveolar bone is used as a tool to move and adjust
the position of teeth [9,10]. Thus, understanding theeffects and mechanisms of mechanical loading on bonecells is important for successful treatment of unloadingrelated osteoporosis, effective orthodontic treatment,
orthopedic surgery, and physical therapy.
Mechanical loading induces multiple cellular eventsin osteoblasts. A major effect of mechanical loading on
osteoblasts is the efflux of adenosine triphosphate(ATP) into the extracellular space [11–14]. Extracellular
ATP can then subsequently act as a signaling moleculein an autocrine or paracrine manner, to activate members of the P2 class of extracellular nucleotide receptors.
Purinergic receptor P2X1 (P2X1R), purinergic receptor P2X7 (P2X7R), and purinergic receptor P2Y2
(P2Y2R) have been reported to be involved in bone
remodeling [11–18]. Variations in P2 receptor subtype
expression and nucleotide ligand responsiveness, however, mean that the net effect of extracellular nucleotides on osteoblast differentiation is unclear. For
example, whereas intermittent compressive force applied
to human mandibular bone-derived osteoblast cells
releases ATP, activates P2X1R, and promotes osteoblast differentiation [14], the activation of P2X7R by
the selective agonist BzATP suppresses the differentiation of primary human osteoblasts [15]. It has also beenreported that low concentrations of ATP and UTPstrongly inhibit osteogenesis in osteoblasts via theP2Y2R [16]. Furthermore, extracellular ATP can bemetabolized into ADP and adenosine by the action ofectonucleotidases and ATP can also act as a source ofphosphate for osteoblast mineralization. Despite thelarge body of evidence that extracellular ATP affects
osteoblast activity, little is known about the mechanism
by which ATP is secreted.
Solute carrier family 17 member 9 (SLC17A9) was
recently identified as a vesicular nucleotide transporter(VNUT), essential for ATP secretion from adrenal
PC12 cells [19]. ATP can be released from cells via multiple mechanisms including through pannexin or connexin hemichannels [20], maxi-anion channels [21],P2X7R [22], and vesicular exocytosis [23]. VNUT localizes to secretory vesicle membranes and is involved in
the efflux of ATP via vesicular exocytosis. That is,
VNUT transports ATP from the cytosol into the secretory vesicle from where ATP can subsequently be exocytosed upon stimulation. So far, VNUT expression has
been reported in various cells such as odontoblasts,

VNUT regulates osteoblastogenesis

periodontal ligament cells, limbal epithelial cells, conjunctival epithelial cells, and gastric epithelial cells[24–28]. Given the importance of extracellular ATP inosteoblast cell function, we hypothesized that VNUT
may play a role in osteoblast activity.
Here, we report that VNUT is expressed in osteoblasts where it plays a role in ATP secretion as well as
osteoblast differentiation.

Materials and methods
Cell culture
MC3T3-E1 cells, a mouse calvarial osteoblast cell line, wereobtained from Riken Bio Resource Center (Tsukuba,
Japan). MC3T3-E1 cells were maintained in α-minimal
essential medium (12571-063; Gibco-BRL, Grand, Island,
NY, USA) supplemented with 10% FBS (Biosera Nuaillé,France) and 100 UmL−1 penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
Osteoblast differentiation was induced by culturing cells inan osteogenic medium containing 50 μgmL−1 ascorbic acid
(FUJIFILM Wako Pure Chemical Corporation) and10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO,
USA) for 7 days. Cells were treated with 1, or 10 μM clodronate (Sigma-Aldrich). Ionomycin (AdipoGen Life
Sciences, San Diego, CA, USA) treatments were performed
at 1 μM. Brefeldin A (Cayman Chemical, Ann Arbor, MI,
USA) was used at 10 μM. HEK293T cells (Takara Bio,
Shiga, Japan) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (12320-032; Gibco-BRL) supplemented
with 10% FBS (Biosera Nuaillé) and 100 UmL−1 penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation).

Application of mechanical loading
Compressive loading by centrifugation was previously
described [29]. Briefly, cells were seeded at a density of 3 × 105
cells per well in 12-well plates. Medium was changed into 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered DMEM without bicarbonate (12320-032; Gibco-BRL),
supplemented with 18 µM of hydrochloric acid (Nacalai Tesque Inc., Kyoto, Japan) to stabilize pH and then cells placedin an incubator (Yamato Scientific Co. Ltd., Tokyo, Japan) at
37 °C for 1 h. The plates were subsequently centrifuged at
4.4 × 10−2 Ncm−2 (5.0 gcm−2) or 8.8 × 10−2 Ncm−2(9.0 gcm−2) using a PlateSpin II centrifuge (Kubota Corp.,
Tokyo, Japan) in the incubator at 37 °C for 12 h. Compressive force by weights was also previously described [30].
Briefly, a thin glass plate was placed unto the cells and a forceof 8.8 × 10−2 Ncm−2 was then applied for 12 h by placing aload unto the glass plate.

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1613

A. Inoue et al.

VNUT regulates osteoblastogenesis

Measurement of extracellular ATP
Extracellular ATP was measured in medium samples
collected from cells using an ATP assay kit (TOYO B-Net,
Tokyo, Japan) according to manufacturer’s instructions[24] and an AB-2270 luminometer (ATTO Corp., Tokyo,
Japan).

RNA extraction, reverse transcription, andquantitative real-time PCRHeart, liver, kidney, skeletal muscle, white adipose tissue,
testis, tongue, bone, and small intestine were obtained from
10-week-old male C57BL/6J mice. Total RNA was isolated
from each organ using a RNAqueous Kit (Ambion; Life
Technologies, Austin, TX, USA) and then reverse-transcribed into cDNA using SuperScript VILO Master Mix(Thermo Fisher Scientific, Waltham, MA, USA). cDNA
was amplified by quantitative real-time PCR (qPCR) using
specific primers for Slc17a9 (forward, AATCCTCACCGGTCTGCTC; reverse, AAAGGCTCTCTCGC
TCTCCT: NM_183161), Collagen 1a1 (forward, GGTATGCTTGATCTGTATCTG; reverse, TCTTCTGAGTTTG
GTGATACG: NM_007742), Runx2 (forward, TTCAACGATCTGAGATTTGTGGG; reverse, GGATGAGGAATGCGCCCTA: NM_001146038), Osterix (forward, AGAGATCTGAGCTGGGTAGAGG; reverse, AAGAGAGCCTGGCAAGAGG: NM_130458), Alkaline phosphatase(Alp) (forward, CGGGACTGGTACTCGGATAA; reverse,
ATTCCACGTCGGTTCTGTTC: NM_007431), P2x1r(forward, CATGGGGACAGCTCCTTTGT; reverse, GA
GTGCAGCCACTGTCATCT: NM_008771), P2x7r (forward, TGCAGCTGGAACGATGTCTTG; reverse, CGCTGGTACAGCTTATCGCTCA: NM_011027), P2y2r (forward, TCAAACCGGCTTATGGGACC; reverse, TCAAA
CCGGCTTATGGGACC: NM_002564), or β-actin (forward, AAGGCCAACCGTGAAAAGAT; reverse, GTGGTACGACCAGAGGCATAC: NM_007393), and PowerUp
SYBR Green Master Mix (Thermo Fisher Scientific) with a
QuantStudio 3 thermal cycler (Thermo Fisher Scientific).
The following cycling parameters were used: 40 cycles of
15 s denaturation at 95 °C and 60 s annealing/extension at
60 °C. Values were normalized to β-actin using the 2ΔΔCt
method [31].

Immunohistochemistry
Animal experiments were reviewed and approved by the
Kyushu Dental University Animal Care and Use Committee (#18-003). Wild-type mouse pups on a C57BL/6J
genetic background were sampled at postnatal day 5. Calvaria bones were fixed with 4% paraformaldehyde (Merck
KGaA, Darmstadt, Germany) in PBS, dehydrated throughan ethanol series, embedded in paraffin, and cut into 4-µm
frontal sections [32]. Immunostaining was performed with

1614

anti-VNUT rabbit polyclonal antibody (1 : 50 dilution).
This antibody against rat or mouse VNUT was generated
in rabbits using synthetic peptides corresponding to residues 5–19, RSSLMQPIPEETRKT [24] or anti-VNUT guinea pig polyclonal antibody (1 : 50 dilution, ABN83;
Sigma-Aldrich), biotinylated anti-rabbit IgG (1 : 400 dilution, PK-6101; Vector Laboratories, Burlingame, CA,
USA) or anti-Guinea pig IgG HRP (1 : 100 dilution,
NB7398; Novus Biologicals, Centennial, CO, USA), andthe Vectastain Elite ABC kit (1 : 50 dilution; Vector Laboratories), Sigmafast 3,30 -diaminobenzidine (DAB) tablets(Sigma-Aldrich) were used for visualization of reaction
products. Immunostained sections were counterstained withdiluted hematoxylin. As a negative control for anti-VNUT
rabbit polyclonal antibody, antibody was preadsorbed withthe antigenic peptide by mixing with 10 µgµL−1 peptide for
60 min at room temperature [24].

Plasmid and transfection
Murine Slc17a9 (NM_183161) was obtained by standard
PCR cloning from mouse white adipose tissue cDNA using
PrimeSTAR HS DNA polymerase (TaKaRa, Otsu, Japan)
and subcloned into pcDNA3.1His-V5 (Thermo Fisher Scientific). Cells were transfected with Slc17a9 plasmid using
Lipofectamine 2000 (Thermo Fisher Scientific) according to
the manufacturer’s instructions.

Immunocytochemistry
Cells were seeded at a density of 2 × 104 cells per well in
96-well plates. After experimental treatment such as compressive loading or transfection, cells were fixed with 4%paraformaldehyde for 10 min and then blocked/permeabilized with PBS containing 0.3% Triton X-100 (FUJIFILM
Wako Pure Chemical Corporation) and 5% goat serum(Gibco-BRL) for 30 min at room temperature. Cells werethen incubated with anti-VNUT rabbit polyclonal antibody(1 : 100 dilution) [24], anti-VNUT guinea pig polyclonal
antibody (1 : 100 dilution, ABN83; Sigma-Aldrich), or
anti-V5 antibody mouse monoclonal antibody (1 : 100 dilution, M167-3, MBL) for 1 h at room temperature. Following incubation with an Alexa 488-conjugated secondary
antibody (1 : 1000 dilution; Thermo Fisher Scientific), cells
were imaged with an ABZ-9000 microscope (Keyence,
Osaka, Japan). To visualize the cell nuclei, the cells were
stained with DAPI (1 : 1000 dilution; Vector laboratories).
To visualize the cytoskeleton, the cells were stained with
Rhodamine Phalloidin (1 : 1000 dilution; Thermo Fisher
Scientific). The fluorescence intensity per unit area of thecells was quantified using IMAGEJ (National Institutes of
Health, Bethesda, MD, USA) [33] . All experiments were
performed at least three independent times. All images wereacquired at the same exposure and contrast settings, andrepresentative images are shown.

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Inoue et al.

VNUT regulates osteoblastogenesis

Measurement of ALP activity
Cells were seeded at a density of 3 × 10 cells per well in
12-well plates. After 7-day treatment with osteoblast differentiation medium, cells were washed with PBS and lysed byfreeze–thaw lysis in 0.1% Triton X (FUJIFILM Wako
Pure Chemical Corporation). Cell lysates were then incubated with a substrate solution of 1.5 mM MgCl2 (pH 10),50 mM NaHCO₃/Na₂CO₃, 1 mgmL−1 p-nitrophenyl phosphate, and absorbance (O.D.) was measured at 405 nm
using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) [34].
5

Short hairpin RNA (shRNA) gene silencing
Vectors expressing shRNAs targeting Slc17a9 (sh-Vnut,
TRCN0000102020, TRCN0000102021, TRCN0000102022)
and the nontargeted control shRNA (sh-scr; NSHMC016)
were purchased from Sigma-Aldrich. For stable silencing of
Slc17a9, lentiviral particles were prepared by transfecting
HEK-293T cells with shRNA vector, psPAX2 viral packaging vector (a gift from D. Trono; Addgene plasmid#12260), and pMD2. G viral envelope encoding vector (agift from D. Trono; Addgene plasmid #12259), using Lipofectamine 2000 (Thermo Fisher Scientific). Lentivirus wascollected 48 h post-transfection and filtered using a 0.45-μm
filter (Merck KGaA). Viral infection of MC3T3-E1 cells
was performed with 8.0 μgmL−1 hexadimethrine bromide
for 24 h. Stable cells were then selected by culturing in thepresence of 5.0 μgmL−1 puromycin (FUJIFILM Wako
Pure Chemical Corporation) for 3 days.

GCUGUGUCCUAACUUCGU) (Thermo Fisher Scientific) or P2y2r (Stealth siRNA, forward, CCCUGC
CGCUGUUGGUUUAUUACUA; reverse, UAGUAAUAAACCAACAGCGGCAGGG) was transfected into
MC3T3-E1 cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the
manufacturer’s protocol.

Statistical analysis
Comparisons were made using unpaired t-test, one-way
analysis of variance and using the Bonferroni method or
Kruskal–Wallis method tests. The results are shown as themean  SD. The statistical significance is indicated as follows: *P < 0.05 and **P < 0.01.

Results
VNUT is expressed by osteoblasts
Expression of Slc17a9 in various murine tissues was
examined by qPCR. mRNA levels of Slc17a9 wereparticularly high in bone and white adipose tissue(Fig. 1A). Immunohistochemical analysis revealed that
VNUT was expressed in osteoblasts within alveolarbone (Figs 1B and S1A). The specificity of antibody
against VNUT was confirmed using negative and positive controls (Fig. S1B-D).

siRNA knockdown

Mechanical loading increases VNUT expression
levelssiRNA against P2x7r (Stealth siRNA, forward, ACGAAGUUAGGACACAGCAUCUUUG; reverse, CAAAGAU

Since mechanical force stimulates ATP release inosteoblasts, we next examined whether compressive

Fig. 1. VNUT is expressed in osteoblasts. (A) The mRNA levels of Slc17a9 in various murine tissues were determined by qPCR. (B)Mandibular bone from 3-day-old C57BL/6J mice was immunostained with rabbit anti-VNUT antibody. The boxed areas in the left panel are
shown as magnified images in the right panel. Scale bars indicate 100 μm (left panel) and 20 μm (right panel), respectively. Arrowheads inthe right panel indicate VNUT-positive osteoblasts. Data are expressed as the mean  SD (n = 3). heart, H; liver, L; kidney, K; skeletal
muscle, SM; white adipose tissue, W; testis, TE; tongue, TO; bone, B; small intestine, SI.

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1615

A. Inoue et al.

VNUT regulates osteoblastogenesis

force on MC3T3-E1 cells affects Slc17a9 expression.
4.4 × 10−2 or 8.8 × 10−2 Ncm−2 loading for 12 h
upregulated the mRNA levels of Slc17a9 in load-dependent manner in MC3T3-E1 cells (Fig. 2A). Under
these conditions, there was no obvious cytotoxicity tocells as assessed by LDH assay (data not shown). The
increase in Slc17a9 expression after 12 h of 8.8 ×
10−2 Ncm−2 compressive force was correlated with anincrease in extracellular ATP (Fig. 2B). To further
assess the role of mechanical loading on Slc17a9
expression, we used an alternate weight-induced
mechanical loading model to apply compressive force
to MC3T3-E1 cells. 8.8 × 10−2 Ncm−2 of force for
12 h led to a significant upregulation of Slc17a9
mRNA levels (Fig. 2C). Furthermore, immunostaining
showed that compressive force increased the fluorescence intensity of VNUT staining (Fig. 2D,E). As with
the immunohistochemical staining, the specificity ofantibody against VNUT was confirmed using negative/
positive control experiments (Fig. S2).

Mechanical loading inhibits osteoblast
differentiation
We next examined the effect of mechanical loading-induced VNUT on osteoblast differentiation. MC3T3-E1
cells were mechanically loaded by centrifugation andthen stimulated to differentiate by the addition of
ascorbic acid and β-glycerophosphate. As shown in
Fig. 3A-D, compressive force suppressed the expression of osteoblast differentiation markers such as
Runx2 (Fig. 3A), Osterix (Fig. 3B), Collagen 1a1(Fig. 3C), and Alp (Fig. 3D). In addition, ALP activity, an indicator of osteoblast function, was also
decreased following compressive force applied by centrifugation (Fig. 3E) or by weight loading (Fig. 3F).

Fig. 2. Compressive force stimulates expression of Slc17a9. (A) mRNA levels of Slc17a9 in MC3T3-E1 cell exposed to 0, 4.4 × 10−2, or
8.8 × 10−2 Ncm−2 of compressive force by centrifuge for 12 h. (B) Extracellular ATP levels in MC3T3-E1 cell exposed to 8.8 × 10−2 Ncm−2of compressive force by centrifuge for 12 h. (C) mRNA levels of Slc17a9 in MC3T3-E1 cells exposed to 8.8 × 10−2 Ncm−2 compressive
force by weights for 12 h. (D) MC3T3-E1 cells treated with or without 8.8 × 10−2 Ncm−2 compressive force by centrifuge for 12 h were
stained with guinea pig anti-VNUT antibody, rhodamine phalloidin, or DAPI. Scale bars indicate 100 μm. (E) The fluorescence intensity of the
VNUT staining shown in (D) was quantified by IMAGEJ. control, C; compressive force by centrifuge, F; compressive force by weights, F(W).
Data are expressed as the mean  SD (n = 3). Statistical analysis was performed with one-way analysis of variance followed by Bonferroni
method (A) and unpaired t-test (B, C, E). *P < 0.05 or **P < 0.01 versus control.

1616

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Inoue et al.

VNUT regulates osteoblastogenesis

Alp

1.4
1.2
1
0.8
0.6
0.4
0.2
0

**

E

**
C

F

ALP activity

VNUT inhibits osteoblast differentiation
To determine whether VNUT affects osteoblast differentiation, we stably knocked down Slc17a9 in
MC3T3-E1 cells using shRNA (Fig. 4A). Extracellular
ATP levels were decreased in Slc17a9-knockdown cells
after mechanical loading (Fig. 4B). In the absence of
mechanical loading, extracellular ATP levels were
unchanged (Fig. S3). In response to osteogenic medium, Slc17a9-knockdown cells displayed enhanced
osteoblast differentiation, as indicated by a higher
expression of osteoblast differentiation marker mRNA
expression (Fig. 4C–E) and increased ALP activity(Fig. 4F) under the mechanical loading condition. In
order to confirm the role of VNUT and exocytosis in
ATP release, we next treated cells with clodronate, a
VNUT inhibitor [35], in combination with ionomycin
and brefeldin A. Ionomycin is a selective calcium ionophore that rapidly induces an increase in intracellular
calcium leading to ATP release. Brefeldin A is an inhibitor of vesicular exocytosis. As shown in Fig. 4G, clodronate and brefeldin A both suppressed release of
ATP from MC3T3-E1 cells suggesting that ATP
release is dependent on VNUT activity and exocytosis.
Clodronate decreased extracellular ATP levels in aconcentration-dependent manner (Fig. 4H) andincreased ALP activity in response to mechanicallyloading (Fig. 4I). These observations were independentof changes in cell number since clodronate did notaffect proliferation of MC3T3-E1 cells (Fig. S4).

F

1.2
1
0.8
0.6
0.4
0.2
0

*

C

0.3

0.3
0.25

0.2
0.15
0.1

**

0.05

F

F

ALP activity

0.25

0

C

Col 1a1C

Relative mRNA levels

F

1.2
1
0.8
0.6
0.4
0.2
0

O.D. 405 nm

C

Osterix

B

O.D. 405 nm

Relative mRNA levels*

D

Relative mRNA levels

Fig. 3. Compressive force suppresses
osteoblast differentiation. (A–E) MC3T3-E1
cells were treated with osteoblast
differentiation medium for 7 days after theapplication of compressive force(8.8 × 10−2 Ncm−2, 12 h), and mRNAlevels of the indicated genes were
determined by qPCR. (E) ALP activity incells treated as in A-E (F) ALP activity in
MC3T3-E1 cells treated with osteoblast
differentiation medium for 7 days after theapplication of 8.8 × 10−2 Ncm−2compressive force by weight for 12 h.
control, C; compressive force by
centrifuge, F; compressive force by
weights, F(W). Data are expressed as themean  SD (n = 3). Statistical analysis
was performed with unpaired t-test.
*P < 0.05 or **P < 0.01 versus control.

1.2
1
0.8
0.6
0.4
0.2
0

Relative mRNA levels

Runx2

A

0.2
0.15

**

0.1

0.05
0

C

F

C

F(W)

Compressive force stimulates P2 receptor
expression and regulates osteoblast
differentiation via P2 receptors
To explore potential mechanisms downstream of
mechanical loading-induced VNUT, we next sought todetermine the role of P2 receptors. Among the P2
receptor family, P2X1R [14], P2X7R [12,13,15,18], andP2Y2R [11,16,17] have been reported to be involved inosteoblastogenesis. P2x7r and P2y2r were highly
expressed in MC3T3-E1 cells relative to P2x1r (Fig. 5
A). Expression levels of P2x7r (Fig. 5B) and P2y2r(Fig. 5C) were increased by compressive loading.
Transfection of siRNA against P2x7r (Fig. 5D) or
P2y2r (Fig. 5E) significantly reduced the expression
levels of P2x7r and P2y2r in MC3T3-E1 cells. Knockdown of P2x7r and P2y2r canceled the suppression of
osteoblast differentiation caused by mechanical loading(Fig. 5F-H,I-K).

Discussion
The data presented in this study reveal a role for
VNUT in osteoblast differentiation and function. We
show that VNUT is expressed in bone and in osteoblasts, and we present evidence that VNUT inhibits
osteoblast differentiation in response to compressive
force. Furthermore, we report that VNUT actions arelinked to ATP release and P2X7R and/or P2Y2R
activity (Fig. 6).

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1617

A. Inoue et al.

VNUT regulates osteoblastogenesis

0.2

**

0.1
0

Scr sh

G

sh

1.5
1
0.5
0

**

**

**

2

1
0

Ion
BfA
Clo

-

+ + + +
- + - +
- - + +

Scr sh

1.4
1.2
1
0.8
0.6
0.4
0.2
0

H

*

I

1

*

0.6

0
μM : 0

2.5
2

1

10

**

1.5
1
0.5
0

Scr shScr shATP

1.2

Relative mRNA levels

2

ATP
3

Relative mRNA levels

Scr*

Col 1a1

E

O.D. 405 nm

ALP activity
0.3

**

2.5

Osterix

D

Relative ATP levels

Scr shRelative mRNA levels

**

14
12
10
8
6
4
2
0

Runx2

C

ATP

Relative ATP levels

Relative mRNA levels
O.D. 405 nm

F

1.2
1
0.8
0.6
0.4
0.2
0

B

Relative ATP levels

Slc17a9

A

ALP activity

**

0.8

**

0.6
0.4
0.2
0
μM : 0

1

10

Fig. 4. Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3-E1 cells stably
expressing control scrambled shRNA or shRNA against murine Slc17a9. (B) Extracellular ATP in MC3T3-E1 cells stably expressing control
scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10−2 Ncm−2, 12 h). (C-E) MC3T3E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium
for 7 days after the application of compressive force by centrifuge (8.8 × 10−2 Ncm−2, 12 h) and mRNA levels of the indicated osteoblast
marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3-E1 cells treated with 1 μM
ionomycin, 10 μM brefeldin A, or 10 μM clodronate for 30 min. (H) MC3T3-E1 cells were exposed to compressive force by centrifugation(8.8 × 10−2 Ncm−2, 12 h) and treated with 0, 1.0, or 10 μM clodronate after which extracellular ATP was measured. (I) MC3T3-E1 cells weretreated with 0, 1.0, or 10 μM clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after theapplication of compressive force by centrifuge (8.8 × 10−2 Ncm−2, 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9, sh;ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean  SD (n = 3). Statistical analysis was performed with
unpaired t-test (A-F) and one-way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). *P < 0.05
or **P < 0.01 versus scramble (A–F) or control (H and I).

Our previous work on VNUT showed its importance in the function of odontoblasts, a mineral-forming cell type similar in many ways to osteoblasts [24].
In addition, we have found VNUT expression in periodontal ligament cells [25] and dental pulp cells [24].
VNUT expression has also been observed in various
other tissues such as biliary epithelial cells [36] andintestinal L cells [37]. Thus, VNUT is a largely ubiquitous nucleotide transporter, but its expression may be
specially adapted in each cell type to metabolic
demands and requirements for ATP after specific stimuli. More specifically, in osteoblasts we observed that
VNUT expression is regulated by mechanical force.
Regulation of VNUT by mechanical stimuli is consistent with our previous observations in human1618

periodontal ligament cells [25] where VNUT expression increased with the application of compressive
force. Other reports suggest that hyperosmotic stressincreases VNUT expression in human corneal andconjunctival epithelial cell lines and could be involvedin ATP release at the ocular surface [38]. In dental
pulp cells however, VNUT expression was regulatedby heat stimulation [24]. In our preliminary data, heatstimulation also increased VNUT expression in
MC3T3-E1 cells (data not shown). It has beenreported that heat stress controls osteoblast differentiation [39–41], so it is also possible that VNUT is
involved in the effects of heat stimulation on osteoblast differentiation. In gastric epithelial cells, VNUT
is involved in ATP release induced by activation of

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Inoue et al.

VNUT regulates osteoblastogenesis

200
100
0

**

0.2
0

Alp

*

1
0.5
0

C

F

1
0.6
0.4

**

0.2
0

Scr

1
0

C

Runx2
1.5
1
0.5
0

F

F

Runx2
1.5

*

1
0.5
0

C

*

1.5
1
0.5
0

C

1.5
1
0.5
0

F

F

Alp

K

2

C

2

F

Osterix

J

Osterix

G

2

si-y2

2

C

2

F

0.8

I

2
1.5

P2y2r
1.2

si-x7

3

F

Relative mRNA levels

0.6
0.4

C

Relative mRNA levels

1
0.8

H

0

E

1.2

Scr

1

y2

P2x7r

D
Relative mRNA levels

x7

2

*

4

Relative mRNA levels

300

*

3

5

Relative mRNA levels

400

4Relative mRNA levels

500

P2y2r

C

Relative mRNA levels

Relative mRNA levels

600

x1

Relative mRNA levels

P2x7r

B

Relative mRNA levels

Relative mRNA levels

A

2
1.5
1
0.5
0

C

F

Fig. 5. P2X7 and/or P2Y2 receptors are involved in the suppressive effect of osteoblast differentiation by compressive force. (A) mRNAlevels of P2x1r, P2x7r, and P2y2r in proliferating MC3T3-E1 cells. (B and C) mRNA levels of P2x7r and P2y2r were increased after theapplication of compressive force by centrifugation (8.8 × 10−2 Ncm−2, 12 h). (D) Cells were transfected with scramble siRNA or siRNAagainst P2x7r and mRNA levels of P2x7r determined 24 h after transfection. (E) Cells were transfected with scramble siRNA or siRNAagainst P2y2r and mRNA levels of P2y2r determined 24 h after transfection. (F-H) Cells transfected with siRNA against P2x7r were treatedwith osteoblast differentiation medium and mRNA levels of the indicated osteoblast marker genes determined 7 days after application ofcompressive force by centrifuge (8.8 × 10−2 Ncm−2, 12 h). (I–K) Cells transfected with siRNA against P2y2r were treated with osteoblast
differentiation medium and mRNA levels of the indicated osteoblast marker genes determined 7 days after application of compressive force
by centrifuge (8.8 × 10−2 Ncm−2, 12 h). P2x1r, x1; P2x7r, x7; P2y2r, y2; control, C; compressive force by centrifuge, F; scrambled siRNA,
Scr; siRNA against P2x7r, si-x7; siRNA against P2y2r, si-y2. Data are expressed as the mean  SD (n = 3). Statistical analysis was
performed with unpaired t-test. *P < 0.05 or **P < 0.01 versus control (B, C, F–H, I–K) or scramble (D and E).

transient receptor vanilloid 4, a nonselective cation
channel activated by mechanical stimulation, osmoticpressure, heat, or chemicals [26]. Our observation that
VNUT expression correlates with extracellular ATP
levels is consistent with findings in odontoblasts andperiodontal ligament cells as well [24,25].
Here, in this study, we report that VNUT inhibits
osteoblast differentiation. Given that ATP has also
been linked to osteoblast differentiation [11–18], it iseasy to speculate that VNUT-induced ATP release

contributes to the inhibition of differentiation. Indeed,treatment of MC3T3-E1 cells with clodronate, an inhibitor of vesicular ATP release, also enhanced osteoblast differentiation. VNUT decreases neurogenesis in
N2a murine neuroblastoma cells [42]. Both neuroblasts
and osteoblasts belong to mesenchymal lineage cells.
We also noted that VNUT was highly expressed inwhite adipose tissue (Fig. 1A). Perhaps VNUT may
regulate the differentiation of other mesenchymal lineage cells, such as adipocytes as well.

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1619

A. Inoue et al.

VNUT regulates osteoblastogenesis

Compressiveforce

ATP release
P2X7R/P2Y2R

VNUT
Inhibit
ATP secretory
vesicleOsteoblast
differentiation

Fig. 6. Model for the suppressive effect of compressive force on
osteoblast differentiation. Compressive force increases extracellular
ATP via induction of VNUT. Consequently, ATP regulates
osteoblast differentiation through the P2X7 and/or P2Y2 receptor.

In this study, the application of compressive force
by centrifugal force suppressed osteoblast differentiation. Kariya et al found that applying tension force toMC3T3-E1 cells increased Runx2 and Osterix expression, extracellular matrix proteins, and ALP activity
and promoted calcium mineral formation [12]. A studyof low-intensity pulsed ultrasound stimulation of
MC3T3-E1 cells reported increased expression of
Runx2 and Osterix [13]. On the other hand, in a study
in which osteoblasts collected from mouse mandibles
were subjected to hydrostatic pressure, the productionof inflammatory cytokines and RANKL, an activatorof bone remodeling, was increased [43]. The inconsistency in these results may be due to the fact that thecellular response to mechanical load differs dependingon the type, magnitude, time, and cell used.
Adenosine triphosphate induces various physiological responses through the activation of P2 receptors.
P2 receptors are divided into the P2Y family of G protein-coupled receptors and the P2X family of ligandgated cation channels [44]. Osteoblasts have beenreported to express both P2X and P2Y receptors [45].
Our results showed that compressive force on MC3T3E1 cells increased ATP release and increased expression of P2x7r and P2y2r. Knockdown of P2x7r andP2y2r canceled the suppression of osteoblast differentiation caused by the application of compressive force.
These results support the inhibitory effects of P2X7R
and P2Y2R on bone formation that have been previously reported [15-18].
Taken together, our findings suggest that compressive-induced VNUT regulates osteoblast differentiation, at least in part, via the extracellular ATP-P2X7R
and/or P2Y2R pathway. Unfortunately, we could notreveal the downstream effector(s) of this extracellular

1620

ATP-P2X7R and/or P2Y2R pathway. It has beenreported that P2X7R or P2Y2R are related with osteogenic pathways such as WNT/β-catenin signaling [15]or ERK1/2 signaling [11]. Thus, mechanism of VNUT
function may involve crosstalk with such signaling
pathways. Further research to explore the mechanisms
and implications of these observations may help thedevelopment of new therapeutics for osteoporosis,bone-related diseases, and orthodontic treatment in an
aging society.

Acknowledgements
We thank Dr. Addison for valuable comments andediting our manuscript. This study was supported by
KAKEN Grant (18K09861) to KN-K.

Conflict of interest
The authors declare no conflict of interest.

Data accessibility
Raw data are available from the corresponding author
upon reasonable request.

Author contributions
AI, TS, MN, TM, and SK performed the experiments.
AI, KN-K, KK-G, MM, TS, MI-S, KY, MN, TM,
YT-S, KM, SK, and TK reviewed the intermediate
draft. AI and KN-K designed the study. AI, KN-K,
SK, and TK performed the literature review, prepared
the initial and final versions of the article, and submitted the document.

References
1 Chambers TJ, Evans M, Gardner TN, Turner-Smith A
and Chow JW (1993) Induction of bone formation in
rat tail vertebrae by mechanical loading. Bone Miner 20,
167–178.
2 Hoshi K, Kawaki H, Takahashi I, Takeshita N, Seiryu
M, Murshid SA, Masuda T, Anada T, Kato R, Kitaura
H et al. (2014) Compressive force-produced CCN2
induces osteocyte apoptosis through ERK1/2 pathway.
J Bone Miner Res 29, 1244–1257.
3 Ichimiya H, Takahashi T, Ariyoshi W, Takano H,
Matayoshi T and Nishihara T (2007) Compressive
mechanical stress promotes osteoclast formation
through RANKL expression on synovial cells. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 103,
334–341.

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Inoue et al.

4 Kawaki H, Kubota S, Suzuki A, Suzuki M, Kohsaka
K, Hoshi K, Fujii T, Lazar N, Ohgawara T, Maeda T
et al. (2011) Differential roles of CCN family proteinsduring osteoblast differentiation: involvement of Smadand MAPK signaling pathways. Bone 49, 975–989.
5 Yamaguchi M (2009) RANK/RANKL/OPG duringorthodontic tooth movement. Orthod Craniofac Res 12,
113–119.
6 Shoji-Matsunaga A, Ono T, Hayashi M, Takayanagi H,
Moriyama K and Nakashima T (2017) Osteocyte
regulation of orthodontic force-mediated tooth
movement via RANKL expression. Sci Rep 7, 1–8.
7 Sibonga JD (2013) Spaceflight-induced bone loss: isthere an Osteoporosis Risk? Curr Osteoporos Rep 11,
92–98.
8 Nguyen J, Tang SY, Nguyen D and Alliston T (2013)
Load regulates bone formation and Sclerostin
expression through a TGFbeta-dependent mechanism.
PLoS One 8, e53813.
9 Roberts WE, Goodwin WC and Heiner SR (1981)
Cellular response to orthodontic force. Dent Clin North
Am 25, 3–17.
10 Krishnan V and Davidovitch Z (2006) Cellular,
molecular, and tissue-level reactions to orthodontic
force. Am J Orthod Dentofacial Orthop 129,469.e1–469.e32.
11 Xing Y, Gu Y, Bresnahan JJ, Paul EM, Donahue HJ
and You J (2014) The roles of P2Y2 purinergic
receptors in osteoblasts and mechanotransduction.
PLoS One 9, e108417.
12 Kariya T, Tanabe N, Shionome C, Manaka S, Kawato
T, Zhao N, Maeno M, Suzuki N and Shimizu N (2015)
Tension force-induced ATP promotes osteogenesis
through P2X7 receptor in osteoblasts. J Cell Biochem
116, 12–21.
13 Manaka S, Tanabe N, Kariya T, Naito M, Takayama
T, Nagao M, Liu D, Ito K, Maeno M, Suzuki N and
et al. (2015) Low-intensity pulsed ultrasound-induced
ATP increases bone formation via the P2X7 receptor in
osteoblast-like MC3T3-E1 cells. FEBS Lett 589,
310–318.
14 Sindhavajiva PR, Sastravaha P, Arksornnukit M andPavasant P (2018) Intermittent compressive force
induces human mandibular-derived osteoblast
differentiation via WNT/β-catenin signaling. J Cell
Biochem 119, 3474–3485.
15 Sindhavajiva PR, Sastravaha P, Arksornnukit M andPavasant P (2017) Purinergic 2X7 receptor activation
regulates WNT signaling in human mandibular-derived
osteoblasts. Arch Oral Biol 81, 167–174.
16 Orriss IR, Utting JC, Brandao-Burch A, Colston K,
Grubb BR, Burnstock G and Arnett TR (2007)
Extracellular nucleotides block bone mineralizationin vitro: evidence for dual inhibitory mechanisms

VNUT regulates osteoblastogenesis

involving both P2Y2 receptors and pyrophosphate.
Endocrinology 148, 4208–4216.
17 Hoebertz A, Mahendran S, Burnstock G and Arnett
TR (2002) ATP and UTP at low concentrations
strongly inhibit bone formation by osteoblasts: a novelrole for the P2Y2 receptor in bone remodeling. J Cell
Biochem 86, 413–419.
18 Orriss IR, Key ML, Brandao-Burch A, Patel JJ,
Burnstock G and Arnett TR (2012) The regulation of
osteoblast function and bone mineralisation by
extracellular nucleotides: the role of p2x receptors. Bone51, 389–400.
19 Sawada K, Echigo N, Juge N, Miyaji T, Otsuka M,
Omote H, Yamamoto A and Moriyama Y (2008)
Identification of a vesicular nucleotide transporter. Proc
Natl Acad Sci USA 105, 5683–5686.
20 Spray DC, Ye ZC and Ransom BR (2006) Functional
connexin "hemichannels": a critical appraisal. Glia 54,
758–773.
21 Sabirov RZ and Okada Y (2005) ATP release via anion
channels. Purinergic Signal 1, 311–328.
22 Suadicani SO, Brosnan CF and Scemes E (2006) P2X7
receptors mediate ATP release and amplification ofastrocytic intercellular Ca2+ signaling. J Neurosci 26,
1378–1385.
23 Zhang Z, Chen G, Zhou W, Song A, Xu T, Luo Q,
Wang W, Gu XS and Duan S (2007) Regulated ATP
release from astrocytes through lysosome exocytosis.
Nat Cell Biol 9, 945–953.
24 Ikeda E, Goto T, Gunjigake K, Kuroishi K, Ueda M,
Kataoka S, Toyono T, Nakatomi M, Seta Y, Kitamura
C et al. (2016) Expression of vesicular nucleotide
transporter in rat odontoblasts. Acta Histochem
Cytochem 49, 21–28.
25 Mizuhara M, Kometani-Gunjigake K, Nakao-Kuroishi
K, Toyono T, Hitomi S, Morii A, Shiga M, Seta Y,
Ono K and Kawamoto T (2020) Vesicular nucleotide
transporter mediates adenosine triphosphate release incompressed human periodontal ligament fibroblast cells
and participates in tooth movement-induced
nociception in rats. Arch Oral Biol 110, 104607.
26 Mihara H, Uchida K, Koizumi S and Moriyama Y(2018) Involvement of VNUT-exocytosis in transient
receptor potential vanilloid 4-dependent ATP releasefrom gastrointestinal epithelium. PLoS One 13, 1–13.
27 Moriyama Y, Hiasa M, Sakamoto S, Omote H and
Nomura M (2017) Vesicular nucleotide transporter(VNUT): appearance of an actress on the stage ofpurinergic signaling. Purinergic Signal 13, 387–404.
28 Miras-Portugal MT, Menendez-Mendez A, GomezVillafuertes R, Ortega F, Delicado EG, Perez-Sen R
and Gualix J (2019) Physiopathological role of thevesicular nucleotide transporter (VNUT) in the central
nervous system: relevance of the vesicular nucleotide

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1621

A. Inoue et al.

VNUT regulates osteoblastogenesis

release as a potential therapeutic target. Front Cell
Neurosci 13, 224.
29 Ueda M, Goto T, Kuroishi KN, Gunjigake KK, Ikeda
E, Kataoka S, Nakatomi M, Toyono T, Seta Y and
Kawamoto T (2016) Asporin in compressed periodontal
ligament cells inhibits bone formation. Arch Oral Biol62, 86–92.
30 Kanzaki H, Chiba M, Shimizu Y and Mitani H (2002)
Periodontal ligament cells under mechanical stressinduce osteoclastogenesis by receptor activator of
nuclear factor κB ligand up-regulation via
prostaglandin E2 synthesis. J Bone Miner Res 17,
210–220.
31 Livak KJ and Schmittgen TD (2001) Analysis ofrelative gene expression data using real-timequantitative PCR and the 2(-Delta Delta C(T))
Method. Methods 25, 402–408.
32 Nakatomi M, Quispe-Salcedo A, Sakaguchi M, IdaYonemochi H, Okano H and Ohshima H (2018) Nestin
expression is differently regulated between odontoblasts
and the subodontoblastic layer in mice. Histochem Cell
Biol 149, 383–391.
33 McCloy RA, Rogers S, Caldon CE, Lorca T, Castro A
and Burgess A (2014) Partial inhibition of Cdk1 in G 2phase overrides the SAC and decouples mitotic events.
Cell Cycle 13, 1400–1412.
34 Kokabu S, Nojima J, Kanomata K, Ohte S, Yoda T,
Toru F and Katagiri T (2010) Protein phosphatasemagnesium-dependent 1A-mediated inhibition of BMP
signaling is independent of Smad dephosphorylation. J
Bone Miner Res 25, 653–660.
35 Kato Y, Hiasa M, Ichikawa R, Hasuzawa N,
Kadowaki A, Iwatsuki K, Shima K, Endo Y, Kitahara
Y, Inoue T et al. (2017) Identification of a vesicular
ATP release inhibitor for the treatment of neuropathic
and inflammatory pain. Proc Natl Acad Sci USA 114,
E6297–E6305.
36 Sathe MN, Woo K, Kresge C, Bugde A, Luby-Phelps
K, Lewis MA and Feranchak AP (2011) Regulation ofpurinergic signaling in biliary epithelial cells by
exocytosis of SLC17A9-dependent ATP-enrichedvesicles. J Biol Chem 286, 25363–25376.
37 Harada Y and Hiasa M (2014) Immunological
identification of vesicular nucleotide transporter inintestinal L cells. Biol Pharm Bull 37, 1090–1095.
38 Guzman-Aranguez A, Pérez de Lara MJ and Pintor J(2017) Hyperosmotic stress induces ATP release andchanges in P2X7 receptor levels in human corneal andconjunctival epithelial cells. Purinergic Signal 13,
249–258.
39 Shui C and Scutt A (2001) Mild heat shock induces
proliferation, alkaline phosphatase activity, andmineralization in human bone marrow stromalcells and Mg-63 cells in vitro. J Bone Miner Res 16,
731–741.

1622

40 Norgaard R, Kassem M and Rattan SI (2006) Heatshock-induced enhancement of osteoblastic
differentiation of hTERT-immortalized mesenchymal
stem cells. Ann N Y Acad Sci 1067, 443–447.
41 Dolan EB, Haugh MG, Tallon D, Casey C andMcNamara LM (2012) Heat-shock-induced cellularresponses to temperature elevations occurring duringorthopaedic cutting. J R Soc Interface 9, 3503–3513.
42 Menéndez-Méndez A, Diaz-Hernandez JI and MirasPortugal MT (2015) The vesicular nucleotide
transporter (VNUT) is involved in the extracellular
ATP effect on neuronal differentiation. Purinergic
Signal 11, 239–249.
43 Yamamoto K, Yamamoto T, Ichioka H, Akamatsu Y,
Oseko F, Mazda O, Imanishi J, Kanamura N and Kita
M (2011) Effects of mechanical stress on cytokine
production in mandible-derived osteoblasts. Oral Dis
17, 712–719.
44 Burnstock G (2007) Physiology and pathophysiology ofpurinergic neurotransmission. Physiol Rev 87, 659–797.
45 Hoebertz A, Townsend-Nicholson A, Glass R,
Burnstock G and Arnett TR (2000) Expression of P2
receptors in bone and cultured bone cells. Bone 27,
503–510.

Supporting information
Additional supporting information may be foundonline in the Supporting Information section at the endof the article.
Fig. S1. Immunohistochemistry with VNUT antibodies. Mandibular bone from 3-days-old C57BL/6J mice
was immunostained with (A) VNUT antibody (Guineapig), (B) PBS alone as a negative control, (C) VNUT
antibody (Rabbit) pre-absorbed with VNUT peptide
antigen as a negative control for the Rabbit VNUT
antibody, or (D) VNUT antibody (Rabbit). Arrowheads in the (A) panel and (D) left panel indicate
VNUT positive osteoblasts and odontoblasts respectively. Odontoblasts, used as a positive control, havepreviously been shown to highly express VNUT [24].
Boxed areas in the (C, D) left panel are shown as magnified images in the right panel. Scale bars indicate 20µm (A, B, C right panel, D right panel) and 100 µm(C right panel, D right panel) respectively.
Fig. S2. Immunocytochemistry with VNUT antibodies.
(A) MC3T3-E1 cells are immunostained with Rabbit
VNUT antibody preabsorbed with VNUT blocking antigen peptide (Control) or VNUT antibody (Rabbit). (B)MC3T3-E1 cells stably expressing control scrambled
shRNA or shRNA against murine Slc17a9 were stainedwith VNUT antibody (Rabbit), rhodamine phalloidin,
or DAPI. (C) MC3T3-E1 cells were transfected with V5tagged VNUT and immunostained with VNUT antibody

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Inoue et al.

(Rabbit or Guinea pig), and anti-V5 antibody and DAPI.
Scale bars indicate 100 µm, scrambled shRNA, Scr;shRNA against murine Slc17a9, Sh.
Fig. S3. Knock down of VNUT does not alter extracellular ATP levels in the absence of mechanical force.
Extracellular ATP levels from MC3T3-E1 cells stably
expressing control scrambled shRNA or shRNAagainst Slc17a9. Scrambled shRNA, Scr; shRNAagainst murine Slc17a9, sh. Data are expressed as the

VNUT regulates osteoblastogenesis

mean  SD (n = 3). Statistical analysis was performed
with unpaired t-test. *P < 0.05 or **P < 0.01 versus
control.
Fig. S4. Inhibition of Vnut function does not affectcell proliferation of MC3T3-E1 cells. MC3T3-E1 cells
were plated into 96-well plates and incubated with 0,
1.0, or 10 μM clodronate. Cell proliferation wasassessed on day 1, 2, or 3 using a Cell Counting Kit-8(DOJINDO).

FEBS Open Bio 10 (2020) 1612–1623 ª 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

1623