Journal of Biosciences and Medicines, 2013, 1, 11-15 JBM
http://dx.doi.org/10.4236/jbm.2013.13003 Published Online December 2013 (http://www.scirp.org/journal/jbm/)
PGJIFs, new mitochondrial PGJ2 target factors, regulate
cell proliferation
Makoto Umeda1, Tatsuya Uebi1, Naoya Maekawa1, 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 January 2013
ABSTRACT
Our previous study showed that prostaglandin J2
(PGJ2) interacting factor (PGJIF) was purified and
identified with magnetic nanobeads. Farther analysis
of PGJ2 function shows that PGJ2 inhibits cell proli-
feration and rhodamine 123 incorporation. Using PGJ2-
immobilized nanobeads, two target proteins were also
purified and identified as PGJIF1 and PGJIF2.
PGJIF1 genetic analysis showed that PGJIF1 regu-
lates cell proliferation as well as rhodamine 123 in-
corporation in mitochondria, indicating that PGJIF1
is one of the PGJ2 target proteins. The other target
protein, PGJIF2, changes its intracellular localization
in PGJ2-dependent manner. Using nanobeads tech-
nology, two PGJ2 target factors were purified and
identified.
Keywords: Prostaglandin J2; Nanobeads; Mitochondria
1. INTRODUCTION
The drug target proteins identification is the way of evi-
dence based drug discovery. Small chemical-immobi-
lized nanobeads were developed as purifying target fac-
tor(s) [1-7].
Prostaglandin J2 (PGJ2) family have been reported to
show various kinds of biological activities, including
adipogenesis. The PGJ2 target in adipocyte is peroxi-
some proliferator-activated receptor
γ (PPAR γ ), which
promotes the expression of the crucial genes for adipo-
genesis [8-11].
However the physiological role of this compound in
vivo still remains as intriguing issue. Since PGJ2 exerts
its biological effects at least in part through a reaction
with cellular proteins, the identification of target mole-
cules of PGJ2 may facilitate the understanding of the
diverse biological activities of PGJ2 in vi v o [12].
Using PGJ2-immobilized nanobeads the new PGJ2
target protein (PGJ2 interacting factor, PGJIF) was puri-
fied and identified voltage dependent anionic channel 1
(VDAC1) as PGJIF1 from crude extracts of HEK293
cells using this affinity purification system [3].
Here first we demonstrate the PGJ2 function in cell
culture system. PGJ2 regulates cell proliferation and
does not regulate cell death in low dose (0.1 - 1.0 µM),
which is natural and is not suitable for PPAR
γ interaction
as well as apoptosis [3]. Moreover we found other PGJ2-
function, PGJ2-reduced rhodamine 123 incorporation.
Using PGJ2-immobilzed nanobeads 2 target factors were
effectively purified and identified, PGJIF1 and PGJIF2.
One of the PGJ2 target factor, PGJIF1, regulated cell
proliferation and rhodamine 123 incorporation. Second
target factor, PGJIF2, changed its intracellular localiza-
tion from nuclear to mitochondria by PGJ2 administra-
tion. Taken together multifunctional PGJ2 has at least 2
target factors, PGJIF1 & PGJIF2, regulate cell prolifera-
tion and rhodamine 123 incorporation.
2. MA TERIALS AND METHODS
2.1. Materials
Most potent prostaglandin J2 derivative, 15-deoxy-
∆12,14-PGJ2, was used as prostaglandin in this study
[13-15]. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC), triethylamine, N,N-dimethyl-4-
aminopyridine (DMAP), N,N-dimethylformamide (DMF),
acetic anhydride, dithiothreitol and iodoacetamide were
purchased from Nacalai Tesque (Kyoto, Japan). Ethyle-
neglycol diglycidyl ether (EGDE) was purchased from
Wako Chemicals (Osaka, Japan). Trypsin was obtained
from Promega (Madison, WI, USA). HEK293 and
MC3T3-E1 cells were obtained from American Type
Culture Collection (Manassas, VA, USA). Antibodies of
anti-PHB2 (Upstate) and anti-VDAC1 (31HL, Calbio-
chem) were used.
2.2. Effective Purification and Identification of
PGJIFs
FG beads were prepared as previously described [16].
*Corresponding a uthor.

M. Umeda et al. / Journal of Biosciences and Medicines 1 (2013) 11-15
12
Epoxy groups on FG beads were aminolysed by NH4OH
and coupled to EGDE to produce FGNEGDE beads [17,
18]. Epoxy groups on FGNEGDE beads were amino-
lysed by NH4OH (FGNEGDEN), carboxylated with suc-
cinic anhydride (FGNEGDENS) and activated with NHS
to produce NHS-activated FGNEGDENS beads [19].
NHS-activated FGNEGDENS beads (5.0 mg) were in-
cubated with 15.0 mM PGJ2 in 500 µL of DMSO con-
taining 10 (V/V)% triethylamine at 25˚C for 24 hours.
Unreacted amino groups on the surface of the beads were
masked with acetic anhydride in DMF containing trie-
thylamine at 25˚C for 24 hours. PGJ2-immobilized b eads
were suspended in distilled water and stored at 4 ˚C until
use [2,19]. PGJ2-immobilized beads or control beads
(200 μg) were equilibrated with binding buffer and in-
cubated with 200 L of the whole cell extracts at 4˚C for 4
hours using RT-50 rotator (15 rpm, TAITEC, Saitama,
Japan) [2]. Affinity purified PGJIFs were separated by
SDS-PAGE and gels subjected to silver staining. The
specific protein bands were excised, reduced with 10
mM DTT followed by alkylation with 55 mM iodoace-
tamide. Band slices were digested w ith trypsin ( 12 g/mL)
overnight and desalted with ZipTip C18 (Millipore, MA,
USA). The extracted peptides were then separated via
nano-flow liquid chromatography (LC, Paradigm MS4,
Tokyo, Japan) using a reverse phase C18 column (Magic
C18, AMR, Tokyo, Japan). The LC eluent was coupled to
a micro-ionspray source attached to a LCQ Advantage
MAX mass spectrometer (Thermo Electron Corporation,
MA, USA). All MS/MS spectra were searched using the
TurboSEQUEST algorithm within the BioWorks 3.2
software (Thermo Electron Corporation, MA, USA;
[18])
2.3. Protein-Pr otein Interaction Analysis
Protein-protein interaction in cell line analysis with im-
muno-precipitated (Ip)-Western blotting was performed
as described previously [20].
2.4. Measurement of Cell Numbers and Viability
The cells were seeded at 1 × 105 cells per well in 24-well
plate. These cells were grown in DMEM supplemented
with 5% FBS with or without PGJ2, and cells were har-
vested and counted the number after indicated time. Each
experiment was performed on triplicate cultures. The
values are reported as means + SEM. Statistical signifi-
cance (p < 0.05) was determined by unpaired Student’s
t-test (STATVIEW).
2.5. Immunocytochemistr y
The cells were plated on 35-mm poly-L-lysine-coated
glass-bottomed dishes. After mitochondria were stained
with 200 nM rhodamine 123 (Wako Chemicals) for 30
min at 37˚C, the cells were fixed for 20 minutes at room
temperature with 3.7% paraformaldehyde and 0.4% Tri-
ton X-100 in PBS, and then sequentially incubated with
anti-PHB2 antibody. Fluorescent images were captured
with a fluorescence microscopy (Keyence, Japan) and
analyzed.
3. RESULTS AND DISCUSSION
3.1. PGJ2 Regulates Cell Proliferation and
Rhodamine 123 Incorporation (Figure 1)
Our previous study [3] and Figure 1(A) showed that
PGJ2 inhibited HEK293 cell growth. The PGJ2 concen-
tration was similar to in vivo (10 n - 1 µM, [11]). The
reduction of cell number was not due to the cell death
(Fi g ure 1(B)), indicating th at PGJ2 inhibited cell growth
significantly. Moreover PGJ2 administration reduced
rhodamine 123 incorporation in few hours (Figure 1(C)),
suggesting that cellular PGJ2 target proteins (so called
receptor) are in the mitochondria.
3.2. Identification of PGJ2 Mitochondrial
Receptor PGJIF (Figure 2)
PGJ2 is immediately converted from PGD2 which is
very unstable prostanoid. In serum mainly PGJ2 is de-
tected, suggesting that most of PGJ2 reaches to the target
tissue (or cells). Cellular PGJ2 target factor is known to
be transcriptional factor PPAR
γ [3,8,9], but there is dis-
crepancy. The active PGJ2 concentration for PPAR
γ is
from 10 µM to 1 mM, but in vivo PGJ2 concentration is
from 10 nM to 1 µM, which is one-thousa ndth [3,12].
Using PGJ2-immobilzed nanobeads (Figure 2(A)),
two factors (PGJIF1 and PGJIF2, respectively) are puri-
fied and identified from HEK293 cells. PGJIF1 &
PGJIF2 encode VDAC1 & PHB2, respectively (Figures
2(C) and (D)). In our previous study only PGJIF1/
VDAC1 was purified and identified. Our purification
system improving made new factor purification be poss-
ible. Low affinity PGJ2 target factor PGJIF2/PHB2 was
also identified. Using antibody against VDAC1 and
PHB2, they bound to PGJ2-immobilized beads (Figure
2(E) and [3]).
3.3. PGJIF1 Regulates Cell Proliferation and
Rhodamine 123 Incorporation (Figure 3)
With genetic approach in cells PGJIF1/VDAC1 was
evaluated for cell proliferation and rhodamine 123 in-
corporation. First, PGJIF1/VDAC1 overexpression vec-
tor was synthesized, and confirmed its expression by
Western blotting (Figure 3(A)). Using this expression
vector, PGJIF1/VDAC1 function in HEK293 cells was
evaluated. As expected, PGJIF1/VDAC1 regulates both
cell proliferation and Rhodamine 123 incorporation

M. Umeda et al. / Journal of Biosciences and Medicines 1 (2013) 11-15
13
Figure 1. PGJ2 regulate cell proliferation and rhodamine 123 incorporation. (A)
PGJ2 inhibited cell number significantly in a dose-dependent manner. Values are
expressed as the mean + SEM (n = 5). *means statistically significant with its ve-
hicle control (empty circle with dot line, p < 0.05); (B) Cell death was not regulated
by PGJ2 administration. The cells were cultivated with PGJ2 for indicated days.
The PGJ2 concentration was 0.1, 0.3 and 1.0 µM, respectively. NS means statisti-
cally non-significant with its vehicle control (p > 0.05); (C) PGJ2 administration
downregulates mitochondrial rhodamine 123 (Rh) incorporation.
Figure 2. Purification and identification of PGJIFs. (A) Preparation of PGJ2-immobilized
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 beads. FGNEGDEN beads were then
coupled with carboxyl groups of PGJ2 in DMF containing EDC, triethylamine and DMAP;
(B) Purification of PGJIF1 & 2 as PGJ2 target factor with PGJ2-immobilzed beads: The
cell extract was mixed with nanobeads (control beads; lanes 2 & 3, PGJ2-immobilized
beads; lanes 4 to 7) and separated. Nanobeads bound proteins were eluted by boiling (Bo,
lanes 2, 4 & 6) or PGJ2 (1 mM; lanes 5, 5 mM; lanes 3 & 7). PGJIF1 & 2 were purified and
indicated; (C) and (D) Identification of PGJIF2 & 1: Polypeptides (a-d) were identified by
ion-spray mass spectrometry; (E) PGJIF2 interacted PGJ2-immobilized beads (lanes 4 & 5)
visualized with anti-PHB2 antibody.
(Figures 3(B) and (C)). Comparison with the data from
PGJ2 administration (Figure 1(A)-(C)), the proliferative
and rhodamine 123 incorporation function of PGJIF1/
VDAC1 was opposite to PGJ2 function, suggesting that
PGJ2 is antagonist of PGJIF1/VDAC1 in terms of cell
proliferation and rhodamine 123 incorporation.
3.4. Evaluation of PGJIF2/PHB2 (Figures 4 and 5)
To study of the function of the PGJ2 low affinity receptor
PGJIF2/PHB2, similar genetic approach was done, but
no obvio us re sults were obtained (data not shown).
Both PGJIF1/VDAC1 and PGJIF2/PHB2 are two
components of “Porin” protein complex located in mito-
chondrial membrane [3]. PGJIF2/PHB2 is also known as
transcriptional cofactor REA (Repressor of ER Activity)
located in the nuclear [21]. Intracellular localization of
PGJIF2/PHB2 was analyzed. In normal condition PHB2
locates in mitochondria (Panels A, D & J in Figure 4),
and we found that 0.1% ethanol administration induced
PHB2 nuclear localization from mitochondria (Panels B
& K in Figure 4), suggesting that PGJIF2/P HB2 respond
to some stress [22]. Moreover additional PGJ2 adminis-
tration made PGJIF2/PHB2 located in mitochondria
(Panels C, F & L in Figure 4), indicating that PGJIF2/
PHB2 bound with PGJIF1/VDAC1 in the mitochondria.
The interaction of PGJIF1/VDAC1 and P GJIF2/PHB2 is
induced by PGJ2 with Ip-Western experiment (data not
shown).
Taken together PGJ2 signaling regulates intracellular

M. Umeda et al. / Journal of Biosciences and Medicines 1 (2013) 11-15
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Figure 3. Evaluation of PGJIF1 (VDAC1) on cell number and rhodamine 123 incorpora-
tion. (A) Evaluation of PGJIF1 (VDAC1) over expression vector. PGJIF1 (VDAC1) over
expression vector was transfected and PGJIF1 (VDAC1) proteins were detected by West-
ern blotting with anti-VDAC1 antibody; (B) Significant higher cell growth in the PGJIF1
(VDAC1) over expressed cells (filled triangle). *means statistically significant with its
control (empty circle, p < 0.05); (C) PGJIF1 (VDAC1) over-expressed cells are higher rho-
damine 123 (Rh) incorporation.
Figure 4. Evaluation of PGJIF2
(PHB2). PGJ2 inhibited EtOH-in-
duced PHB2 nuclear localization.
The cells treated with 0.1% EtOH
(panels B, C, E, F, H, I, K & L) and 1
µM PGJ2 (panels C, F, I & L) were
visualized with anti-PHB2 antibody
(red color, panels A-C & J-L), rho-
damine 123 (green color, panels D-
F), DAPI (blue color, panels G-L).
The original size of each panel is 30
µm × 30 µm.
Figure 5. Possible model of PGJ2 action.
New mitochondrial PGJ2 target proteins
(PGJIF1/VDAC1, PGJIF2/PHB2) make
complex in a PGJ2-dependent manner.
EtOH induces dissociation of the com-
plex and PHB2 changes its intracellular
localization from mitochondria (green
color) to nuclear (blue color).
localization of PGJIF2/PHB2 (Figure 5). The PGJ2 re-
gulates cell proliferation through binding to the PGJIF1/
VDAC1 and PGJ I F2/ PHB2.
4. CONCLUSION
The nanobeads technology enable to efficient purify Pro-
staglandin J2 targe t fa c t ors for new drug discovery.
5. ACKNOWLEDGEMENTS
We are grateful to our department members in NCGG for helpful dis-
cussions. This work was supported by a Grant-in-Aid for the Ministry
of Education, Culture, Sports, Science and Technology.
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