Journal of Biosciences and Medicines, 2013, 1, 1-4 JBM
http://dx.doi.org/10.4236/jbm.2013.13001 Published Online December 2013 (http://www.scirp.org/journal/jbm/)
Prohibitins, novel vitamin K2 tar ge t fa ct ors in osteoblast
Tatsuya Uebi1, Makoto Umeda1, Naoya Maekawa1, Satoshi Karasawa2, Hiroshi Handa2, Takeshi Imai1
1Department of Aging Intervention, National Center for Geriatrics and Gerontology (NCGG), Obu, Japan
2Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Received August 2013
ABSTRACT
Vitamin K2 (VK2, menaquinone) is a drug for osteo-
porosis. VK2 acts as a cofactor for γ-glutamyl car-
boxylase, which catalyzes the carboxylation of specific
glutamic acid residues (γ-carboxylation) of substrate
proteins. Here we demonstrate that VK2 also regulate
osteoblastgenic marker gene expression. Using VK2-
immobilzed nanobeads new target proteins were pu-
rified and identified from osteoblastic cell line. They
are prohibitin 1 and 2 (PHB1 & 2), respectively. To
confirm the PHBs function on VK2-dependent tran-
scription, PHB1 & 2 were knock-down and osteocal-
cin gene 2 transcripti ons were analyzed, indicating
that PHBs regulate VK2-dependent transcription.
Taken together PHBs are VK2 target proteins for
osteoblastgenic transcription.
Keywords: Vitamin K2; Prohibitin; Osteoblast;
Runt-Related Transcription Factor 2 (Runx2)
1. INTRODUCTION
Vitamin K (VK) is a fat -so luble vitamin that was discov-
ered in 1929 [1]. There are three types of VK: naturally
occurring VK1 (phylloquinone) and VK2 (menaquinone,
MK) and chemically synthesized VK3 (menadione).
VK2 is also known as MK-n (n = 1 to 14), where n
stands for the number of repeating isoprenyl units in its
side chain [2]. The most common form of VK in animals
is MK-4, which is produced by intestinal bacteria or is
metabolically converted from other VKs [3]. VK was
originally discovered as an essential factor for blood coa-
gulation [4]. VK acts as a cofactor for γ-glutamyl car-
boxylase, which catalyzes the carboxylation of specific
glutamic acid residues (γ-carboxylation) of substrate
proteins. VK-dependent γ-carboxylation plays an impor-
tant role in bone homeostasis. Osteocalcin, a critical reg-
ulator of calcium uptake and bone mineralization in os-
teoblasts, is activated by γ-carboxylation [5]. Vitamin K
deficiency causes bleeding diath esis, particularly in new-
born babies [6]. In addition, undercarboxylation of os-
teocalcin due to vitamin K deficien cy is thought to result
in osteoporosis [7]. Thus, MK-4, one of the most potent
VKs, has been widely used as a therapeutic drug for the
above-mentione d diseases [8].
In all the in vitro study VK2 concentration is generally
too high (10 - 100 μM), suggesting that there is other
VK2 target protein(s) with higher affinity to VK2. So, we
selected osteoblast cell system among several systems,
because VK2 effect was significantly detected in 10 nM
concentration.
2. MA TERIALS AND METHODS
2.1. Cell Culture & Extract
Mouse calvaria-derived osteoblastic cell line MC3T3-E1
[9] was maintained in α-minimal essential medium sup-
plemented with 10% fetal calf serum. Cells were plated
at a density of 1.5 ~ 3 × 106 cells/60 -mm dish and, after
48 hours, re-fed with the same medium supplemented
with 0.5% fetal calf serum. After 24 hours, the cells were
treated with various reagents or an equal volume of ve-
hicle [10,11].
MC3T3-E1 cells were cultivated and harvested. The
cell pellets were washed with PBS several times, and
solubilized with binding buffer (20 mM HEPES-NaOH
pH 7.9, 10% glycerol, 200 mM KCl, 1 mM MgCl2, 0.2
mM CaCl2, 0.2 mM EDTA, 1 mM DTT and 0.2 mM
PMSF) with 1% n-octyl-β-D-glucoside (n-octylglucoside),
and centrifuge 1300 g for 5 minutes and supernatant was
recovered. The supernatant was dialyzed against binding
buffer for 4 hours for elimination of n-octylglucoside.
The nuclear extract was prepared according to the me-
thod of Dignam et al. [12] for immuno-precipitation in te-
raction experiments in nuclear.
2.2. Alkaline Phosphatase Assay
MC3T3-E1 cells were cultivated in 24-well plates. After
reaching confluency, medium was supplemented with 60
μg/mL ascorbic acid and 10 nM dexamethasone and cul-
tured for 7 more days. Cells were harvested and analyzed
their alkaline phosphatase activity using commercial kit
(Reporter Assay kit, SAK-101, TOYOBO) with manu-

T. Uebi et al. / Journal of Biosciences and Medicines 1 (2013) 1-4
2
facturing protocols.
2.3. Luciferase Analysis
The luciferase analysis was performed with manufac-
ture’s (Promega) introduction described previously [13].
Mutated OSE2 site was replaced
5’-gcaatcacc-ACCACA-gcatc-3’ (−137 - −130) to BamHI
site (5’-gcaatcacc-GAATTC-gcatc-3’) in the OG2 pro-
moter [14-16].
2.4. Vectors
Runx2 over expression vector was kindly provided from
Pr Komori [17]. The shRunx2 and shPHB1 vectors were
purchased from SantaCruz. For the expression of
shPHB2 RNA, the mouse U6 promoter (positions −315
to +5) was cloned into pBluescript SK+. A double-
stranded oligonucleotide was inserted downstream of the
promoter so as to express the following RNA; 5’-
CCACAT CACAGAACCGAATCTATC-ttcaagaga-GAT-
AGATTCGGTTCTGTGATGTGG-3’ (942 - 965 bp of
NM_007531.2) .
2.5. VK2-Immobilized Beads
FG be ads were p repared as previously des cribed [13,18].
Epoxy groups on FG beads were aminolyzed by NH4OH
and coupled to ethylene glycol diglycidyl ether (EGDE)
to produce FGNEGDE beads. Epoxy groups on
FGNEGDE beads were aminolyzed by NH4OH to pro-
duce FGNEGDEN beads. FGNEGDEN beads (5.0 mg)
were incubated with 5.0 mM VK2 in 500 μL of DMF
containing EDC, triethylamine and DMAP at 25˚C for 24
hours. Unreacted amino groups on the surface of the
beads were masked with acetic anhydride in DMF con-
taining triethylamine at 25˚C for 24 hours. VK2-immo-
bilized beads were suspended in distilled water and
stored at 4˚C until use [2,19].
2.6. Statistical Analysis
Values are reported as mean + SEM. Statistical signifi-
cance (*p < 0.05; **p < 0.005; ***p < 0.0001) w as shown.
Non-statistical differences (p > 0.05) were shown as NS
(non-significance).
3. RESULTS AND DISCUSSION
3.1. VK2 Induces Osteoblast Dif f erentiati on
Markers (Figure 1)
VK2 was administrated to the osteoblastic cell line
MC3T3-E1, and its osteoblast differentiation markers
were analyzed. First, alkaline phosphatase (ALP) activity
was analyzed in several doses (~1 μM) of VK2. ALP
activity was significant and 3.5-fold induced in a dose
Figure 1. VK2 induces osteoblast differentiation markers.
VK2 was administrated to osteoblastic cell line MC3T3-
E1, and the cell activities of ALP (a), OG2-luciferase and
dOSE2-luciferase (b) were analyzed. The VK2 concen-
tration was 0 (vehicle), 1, 10, 100 and 1000 nM (A), re-
spectively. Values are expressed as the mean + SEM (n =
5). *p < 0.05; **p < 0.005; ***p < 0.0001.
dependent manner (Figure 1(a)). Similar results were
obtained in the other osteoblast markers of osteocalcin
gene2 (OG2) promoter activities, especially OSE2 site
[13-15], resulting that VK2 induced osteoblast differen-
tiation in transcriptional level. VK2 regulates Rnux2 ac ti-
vity. Runx2 point mutant or heterozygotes results in Clei-
docranial Dysplasia (CCD; [17,20]). Our data showed
that VK2 induces Runx2 activity, suggesting that VK2
have possibility to apply to CCD therap y.
3.2. Pr eparation of VK2-Immobilized Beads
(Figure 2(a))
To purify new target for VK2, we prepared VK2-immo-
bilized beads. A schematic representation of the proce-
dure for conjugating VK2 to FG-beads is depicted in
Figure 2(a). Briefly, epoxy groups on FG beads were
aminolyzed by NH4OH and coupled to EGDE to produce
FGNEGDE beads. EGDE, introduced as a spacer is im-
portant for reduction of steric hindrance. Epoxy groups
on FGNEGDE beads were aminolyzed by NH4OH to
produce FGNEGDEN beads. VK2 was then conjugated
to FGNEGDE N bea d s .
3.3. Purification and Identification of VK2
Ta rget Proteins (Figures 2(b)-(d))
Using VK2-immobilzed nanobeads, new target proteins
were purified from MC3T3-E1 cell extracts directly. LC-
MS analysis showed that 2 protein bands are corres-
ponded to prohibitin 1 and 2 (PHB1 and PHB2), respec-
tively. No polypeptide from Runx2 was obtained, sug-
gesting that VK2 binds to PHBs and regulates Runx2
activity.
3.4. PHBs Regulate VK2-Dependent Runx2
Transcriptional Activity (Figure 3)
PHBs are known as estrogen (E2)-dependent transcrip-

T. Uebi et al. / Journal of Biosciences and Medicines 1 (2013) 1-4
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Figure 2. VK2-immobilized beads preparation and purification
of new VK2 target proteins. (a) Preparation of VK2-immobi-
lized nanobeads. Epoxy groups on FG beads were aminolyzed
by NH4OH (FGN beads) and coupled to EGDE to produce
FGNEGDE beads. Epoxy groups on FGNEGDE beads were
aminolyzed by NH4OH to produce FGNEGDEN be ads. FGNE-
GDEN beads were then coupled with carboxyl groups of 15d-
PGJ2 in DMF containing EDC, triethylamine and DMAP; (b)
Purification of VK2 target proteins from MC3T3 E1 cell ex-
tracts directly. The cell extracts were mixed with VK2-immo-
bilized beads (K2, lane 3) or control beads (Co, lane 2), and
bound proteins were separated by SDS-PAGE (5% - 20% gra-
dient gel) and visualized by silver staining; (c) and (d) Identifi-
cation of new VK2 target proteins. Seven (a-g) and eight (h-o)
polypeptides were identified by ion-spray mass spectrometry.
Identified amino acid sequences are indicated.
Figure 3. PHBs regulate VK2-dependent transcription activity.
(a) Evaluation of PHB2 over expression and knock down vec-
tors. The control vectors (lanes 1 & 2), PHB2 over expression
vectors (lanes 2 & 3) and shPHB2 vectors (lane 3) were trans-
fected to HEK293 FT cells. The cells were extracted and ly-
sates were subjected into SDS-PAGE and Western blotted with
anti-PHB2 antibody; (b) New VK2 target proteins PHBs regu-
late VK2-dependent OG2 transcription. Vehicle (columns 1, 3,
5 & 7) and 1 μM VK2 (lanes 2, 4, 6 & 8) were administrated to
MC3T3-E1 cells. Control (columns 1 & 2), Runx2 (columns 3
& 4), PHB1 (columns 5 & 6) and PHB2 (columns 7 & 8) sh
vectors were introduced to the cells, and analyzed luciferase
activities. Values are expressed as the mean + SEM (n = 5). *p
< 0.05, and NS, p > 0.05 not significant.
tion regulators [21], but Runx2-interaction was not re-
ported. First, PHB2 over expression and knock-down
(KD) vectors were established. They were introduced
into HEK293FT cells, and PHB2 proteins were detected
(Fi g u re 3 (a)). Functions of the 2 vectors were conf irmed.
Using KD vectors, contribution of Runx2 and PHBs on
OG2 transcription was analyzed. Without VK2 (columns
1, 3, 5 & 7) only Runx2 KD reduced significantly OG2
transcription, sugges ting that PHBs do not affect on basal
OG2 activity, but Runx2 regulates basal OG2 transcrip-
tion via OSE2 (Figure 1(b)). In the meanwhile, control
KD vector introduction significantly induced luciferase
activity by VK2 induction (columns 1 and 2), but in the
case of other KD vectors (Runx2 & PHBs) no significant
induction was observed by VK2 administration, indicat-
ing that Runx2, PHB1 & PHB2 are contributed to the
VK2-dependent transcription.
PHB2 was identified as Estrogen Receptor α (ERα)
modulator, and PHB2 regulates not only ERα function,
but also other transcription factors [21], indicating that
one example is Runx2, which we show here, ERα and
other possibility. As next step, other down stream factor
of PHBs will be identified.
4. CONCLUSION
The concentration of VK2 on osteoblastgenesis is lower
than others, less than 1 μM. VK2 induced osteoblastgen-
ic activities. One of the VK2 signaling pathways is me-
diated through the Runx2 activity. To identify the VK2
target protein(s) VK2-immobilized nanobeads were es-
tablished, and 2 proteins, PHB1 and 2, were purified
from osteoblast cell extracts directly. By OG2 reporter
analysis with KD vectors, PHB1 & 2 are responsible for
VK2-dependent OG2-transcription. Taken together, PHB1
& 2 are new VK2 target proteins in osteoblastgenesis.
5. ACKNOWLEDGEMENTS
We thank to Pr Komori for Runx2 vectors. We are grateful to our de-
partment members in NCGG for helpful discussions. This work was
supported by a Grant-in-Aid for the Ministry of Education, Culture,
Sports, Science and Technology.
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