Prohibitins, novel vitamin K2 target factors in osteoblast

doi:10.4236/jbm.2013.13001

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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/)

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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

Email: [email protected]

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

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

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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