PGJIFs , new mitochondrial PGJ 2 target factors , regulate cell proliferation

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 proliferation and rhodamine 123 incorporation. Using PGJ2immobilized nanobeads, two target proteins were also purified and identified as PGJIF1 and PGJIF2. PGJIF1 genetic analysis showed that PGJIF1 regulates cell proliferation as well as rhodamine 123 incorporation 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 technology, two PGJ2 target factors were purified and identified.

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 molecules of PGJ2 may facilitate the understanding of the diverse biological activities of PGJ2 in vivo [12].
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 PPARγ interaction as well as apoptosis [3].Moreover we found other PGJ2function, 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 localization from nuclear to mitochondria by PGJ2 administration.Taken together multifunctional PGJ2 has at least 2 target factors, PGJIF1 & PGJIF2, regulate cell proliferation and rhodamine 123 incorporation.
Epoxy groups on FG beads were aminolysed by NH 4 OH and coupled to EGDE to produce FGNEGDE beads [17,18].Epoxy groups on FGNEGDE beads were aminolysed by NH 4 OH (FGNEGDEN), carboxylated with succinic anhydride (FGNEGDENS) and activated with NHS to produce NHS-activated FGNEGDENS beads [19].NHS-activated FGNEGDENS beads (5.0 mg) were incubated with 15.0 mM PGJ2 in 500 µL of DMSO containing 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 triethylamine at 25˚C for 24 hours.PGJ2-immobilized beads 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 incubated 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 iodoacetamide.Band slices were digested with 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])

Protein-Protein Interaction Analysis
Protein-protein interaction in cell line analysis with immuno-precipitated (Ip)-Western blotting was performed as described previously [20].

Measurement of Cell Numbers and Viability
The cells were seeded at 1 × 10 5 cells per well in 24-well plate.These cells were grown in DMEM supplemented with 5% FBS with or without PGJ2, and cells were harvested and counted the number after indicated time.Each experiment was performed on triplicate cultures.The values are reported as means + SEM.Statistical significance (p < 0.05) was determined by unpaired Student's t-test (STATVIEW).

Immunocytochemistry
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% Triton X-100 in PBS, and then sequentially incubated with anti-PHB2 antibody.Fluorescent images were captured with a fluorescence microscopy (Keyence, Japan) and analyzed.

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 concentration was similar to in vivo (10 n -1 µM, [11]).The reduction of cell number was not due to the cell death (Figure 1(B)), indicating that 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.

Identification of PGJ2 Mitochondrial
Receptor PGJIF (Figure 2) PGJ2 is immediately converted from PGD2 which is very unstable prostanoid.In serum mainly PGJ2 is detected, suggesting that most of PGJ2 reaches to the target tissue (or cells).Cellular PGJ2 target factor is known to be transcriptional factor PPARγ [3,8,9], but there is discrepancy.The active PGJ2 concentration for PPARγ is from 10 µM to 1 mM, but in vivo PGJ2 concentration is from 10 nM to 1 µM, which is one-thousandth [3,12].Using PGJ2-immobilzed nanobeads (Figure 2(A)), two factors (PGJIF1 and PGJIF2, respectively) are purified 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 possible.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]).

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 incorporation.First, PGJIF1/VDAC1 overexpression vector 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

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 obvious results were obtained (data not shown).Both PGJIF1/VDAC1 and PGJIF2/PHB2 are two components of "Porin" protein complex located in mitochondrial 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/PHB2 respond to some stress [22].Moreover additional PGJ2 administration 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 PGJIF2/PHB2 is induced by PGJ2 with Ip-Western experiment (data not shown).

CONCLUSION
The nanobeads technology enable to efficient purify Prostaglandin J2 target factors for new drug discovery.

Figure 1 .
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 vehicle 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 statistically non-significant with its vehicle control (p > 0.05); (C) PGJ2 administration downregulates mitochondrial rhodamine 123 (Rh) incorporation.

Figure 5 .
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 complex and PHB2 changes its intracellular localization from mitochondria (green color) to nuclear (blue color).