Address correspondence and reprint requests to Sun Ah Park, Associate professor of Neurology, Soonchunhyang University Bucheon Hospital, 1174, Jung-dong, Wonmi-gu, Bucheon, Gyeonggi-do, Republic of Korea 420-767. E-mail: firstname.lastname@example.org
Caspase cleavage of amyloid precursor protein (APP) has been reported to be important in amyloid beta protein (Aβ)-mediated neurotoxicity. However, the underlying mechanisms are not clearly understood. In this study, we explored the effect of caspase cleavage of APP on tau phosphorylation in relation to Aβ. We found that Asp664 cleavage of APP increased tau phosphorylation at Thr212 and Ser262 in N2A cells and primary cultured hippocampal neurons. Compared with wild-type APP, protein phosphatase 2A (PP2A) activity was significantly increased when Asp664 cleavage was blocked by the D664A point mutation. Furthermore, we found that over-expression of C31 reduced PP2A activity. C31 binds directly to the PP2A catalytic subunit, through the asparagine, proline, threonine, tyrosine (NPTY) motif, which is essential for C31-induced tau hyperphosphorylation. However, it appears that the other fragment produced by Asp664 cleavage, Jcasp, modulates neither PP2A activity nor tau hyperphosphorylation. Asp664 cleavage and accompanying tau hyperphosphorylation were remarkably diminished by blockage of Aβ production using a γ-secretase inhibitor. Taken together, our results suggest that Asp664 cleavage of APP leads to tau hyperphosphorylation at specific epitopes by modulating PP2A activity as a downstream of Aβ. Direct binding of C31 to PP2A through the C31-NPTY domain was identified as a mechanism underlying this effect.
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
yellow fluorescence protein
Amyloid plaques, which consist of amyloid beta protein (Aβ) and neurofibrillary tangles composed of hyperphosphorylated tau are the hallmarks of Alzheimer's disease (AD). The interaction between these two major pathogenic proteins contributes synergistically to AD progression. The exposure of tau to Aβ accelerates tau pathology, such as hyperphosphorylation (De Felice et al. 2008; Ryan et al. 2009) and mislocalization into dendritic spines, leading to a loss of spines and degeneration of dendrites (Zempel et al. 2010). In addition, Aβ accelerates the spatiotemporal progression of tau pathology, as demonstrated in an AD mouse model (Hurtado et al. 2010). Numerous studies have shown that tau is a downstream mediator of Aβ. However, the mechanism through which Aβ induces tau pathology is not understood.
Previously, it was observed that caspase activation precedes tangle formation by hours to days, and that the cleavage of tau at Asp421 by caspases promotes tangle formation by causing the misfolding of normal tau (de Calignon et al. 2010). Aβ enhances caspase activation in vitro and in vivo (Gamblin et al. 2003; D'Amelio et al. 2011), resulting in synaptic dysfunction and memory decreases in Tg2576 mice (D'Amelio et al. 2011). Therefore, it is possible that caspase activation links Aβ and tau in AD.
Caspases directly cleave amyloid precursor protein (APP) at the Asp664 residue (based on the APP695 sequence), releasing the potentially cytotoxic C-terminal C31 fragment. There has been controversy regarding the pathogenic role of caspase cleavage of APP in AD (Galvan et al. 2006; Harris et al. 2010). However, an APP-D664A mutation blocking the caspase cleavage of APP delayed the AD phenotype, including synaptotoxicity, brain atrophy, and behavioral abnormalities (Galvan et al. 2006; Bredesen et al. 2010). Cleavage at Asp664 is facilitated by Aβ, and C31 mediates Aβ toxicity as a downstream molecule (Lu et al. 2003; Shaked et al. 2006). Considering that caspase activation is a pathway shared by Aβ and tau, it is likely that caspase cleavage of APP is related to tau pathology. However, the interplay between these two pathways has not been explored. We investigated whether the caspase cleavage of APP at Asp664 has an impact on AD-related tau pathology, particularly in terms of phosphorylation.
Materials and methods
The pcDNA–APP–yellow fluorescence protein (YFP) construct, which was kindly provided by Dr. Christoph Kaether (EMBL, Heidelberg, Germany), has been described previously (Kaether et al. 2000). To construct the caspase cleavage mutant (pcDNA-APP-D664A-YFP), aspartate was substituted by alanine at amino acid 664 (APP695 numbering) using a site-directed mutagenesis kit (QuickChange; Stratagene, La Jolla, CA, USA). Green fluorescence protein (GFP) was fused to the C-terminus of C31 or Jcasp to derive the C31–GFP or Jcasp–GFP construct (Park et al. 2009), respectively, by subcloning into pEGFP-N3 (Clontech, Mountain View, CA, USA). Using the C31–GFP construct, we deleted the asparagine, proline, threonine, tyrosine (NPTY) sequence (residues 684–687 of APP695), to generate C31ΔNPTY–GFP.
Reagents and treatment
Antibodies against p-tau (S396: #9632), p-Akt (S473: #9271), total Akt (#9272), p-extracellular signal-regulated kinases (Erk) (#9101), and p-3-phosphoinositide-dependent protein kinase-1 (PDK1) (#3061) were purchased from Cell Signaling (Beverly, MA, USA). Antibodies against cdk5 (sc-173), p-GSK3β (Y216: sc-135653), GFP (sc-9996), and p-PP2A (at Tyr307: sc-12615) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). P-Tau (T212: 44740G), p-tau (T231: 44746G), p-tau (S199: 44734G), and p-tau (S262: 44750G) were obtained from Invitrogen Life Technologies (Rockville, MD, USA). Antibodies against protein phosphatase 2A (PP2A: #05-421) (catalytic subunit) were purchased from Millipore (Billerica, MA, USA). Tau5 (SIG-39413) was obtained from Covance (Berkeley, CA, USA). CT-15 recognizes the C-terminal 15 amino acids of APP, whereas APP-α664 detects the neo epitope at position 664 after cleavage (Park et al. 2009). Antibodies against β-actin, okadaic acid, and DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) were obtained from Millipore.
Cell culture and transfection
Murine N2A neuroblastoma cells cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum were transfected at 70%–80% confluency using Lipofectamine 2000 (Invitrogen Life Technologies). Constructs (4 μg) were transfected into each well of a six-well plate according to the manufacturer's recommendations, and incubated at 37°C for 24 h. For the experiment using different concentrations of GFP-tagged C31 or Jcasp, 4 μg and 1 μg of the DNA constructs were used. The expression of individual constructs was confirmed by western blot analysis using specific antibodies
Hippocampal primary neuronal culture and transfection
Primary rat hippocampal neuronal cell cultures were prepared from 18-day-old Sprague–Dawley rat fetuses after sacrifice of mother rats. Rat hippocampi were dissected and incubated for 25 min at 37°C in a solution of 2 mg/ml trypsin in Hank's balanced salt solution (HBSS) buffered with 10 mM HEPES (Gibco Life Technologies, Grand Island, NY, USA). The tissue was exposed for 5 min to trypsin inhibitor (1 mg/mL in HBSS) and 0.1 mg/mL DNase. The dissociated cells were resuspended in B27-supplemented neurobasal (NB) medium and plated onto coverslips coated with poly-d-lysine (Sigma, St. Louis, MO, USA) in 60-mm culture dishes at approximately 30 × 104 cells per dish. Non-neuronal cell division was arrested after 48 h in 5 μM cytosine arabinoside (Ara-C). Cultured hippocampal primary neuronal cells were incubated for 7 days at 37°C in 5% CO2 in B27-NB medium, and then transfected with 6 μg of plasmid construct using calcium phosphate in transfection medium [minimal essential medium (MEM); 100 mM pyruvate, 20% glucose, 200 mM glutamine, and HEPES] and maintained at 37°C in 5% CO2.
Transfected cells were lysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer [62.5 mM Tris (pH 6.8), 1% SDS, 10% glycerol, 5% β-mercaptoethanol], boiled for 5 min, subjected to SDS–PAGE, and transferred to an Immobilon membrane (Millipore). The membrane was blocked with 5% skim milk in tris-buffered saline tween-20 (TBST) [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1% Tween 20] and then incubated with primary antibodies in blocking buffer overnight. Bound primary antibodies were detected by incubation with horseradish peroxidase-conjugated secondary antibodies (1 : 1000; Santa Cruz Biotechnology) for 1 h, followed by enhanced chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ, USA). The respective protein band intensities were quantified by densitometry using the ImageJ 1.42q software (NIH, Bethesda, MD, USA).
At 24 h post transfection, the hippocampal neuronal cells were washed twice with cold phosphate buffered saline (PBS) and fixed in 4% formaldehyde for 10 min at 4°C. After fixation, the cells were permeabilized in 0.1% Triton X-100/2% bovine serum albumin (BSA) for 5 min at 21°C. Next, the cells were incubated overnight at 4°C with 1 : 200 rabbit anti-p-tau (Thr212) or 1 : 200 rabbit anti-p-tau (Ser262) antibody, washed with PBS three times, and incubated with Alexa Fluor 555 anti-rabbit secondary antibodies (Invitrogen Life Technologies) for 1 h at 21°C. Nuclei were further stained for 30 min with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) in PBS. The stained cells were examined by fluorescence microscopy (Axiovert 200M; Carl Zeiss, Jena, Germany).
PP2A activity assay
PP2A activity was examined using a molybdate dye-based phosphatase assay kit (V2460; Promega, Madison, WI, USA). Transfected cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonylfluoride (PMSF), and Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland)] for 30 min on ice. The samples were then applied to a reaction pre-mix that contained a phosphopeptide substrate, 5 × PP2A reaction buffer [250 mM imidazole (pH 7.2), 1 mM EGTA, 0.1% β-mercaptoethanol, 0.5 mg/mL BSA], and storage buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 0.02% sodium azide] in 96-well plates. After 10 min at 37°C, the reactions were stopped by the addition of a molybdate dye/additive mixture. Absorbance of the supernatant was measured at 630 nm. Phosphatase activity was calculated using a phosphatase standard curve.
Transfected cells were washed with ice-cold PBS and lysed in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM PMSF, and Complete Mini Protease Inhibitor Cocktail] for 30 min on ice. The lysate was centrifuged (10 000 g, 15 min), and the supernatant was transferred to a new tube. For the immunoprecipitation of YFP-tagged or GFP-tagged C-terminal fragments of over-expressed APP, the lysates (2000 μg of protein) were incubated with 1 mg of anti-GFP or anti-PP2A antibody overnight at 4°C. The immunoprecipitates were collected with 20 mL of protein G plus A-agarose for 1 h at 4°C. The immunoprecipitated proteins were resolved by SDS–PAGE, transferred electrophoretically to an Immobilon membrane, and probed using the CT-15 (1 : 500), anti-GFP (1 : 1000), anti-PP2A (1 : 1000), and 6E10 (1 : 1000) antibodies.
Statistical analyses were performed using the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Differences between the means of the vector-, wild-type APP (APPwt)-, and APP-D664A-transfected samples were analyzed by one-way anova with Tukey's post hoc test, with a significance level of p <0.05. Comparison of the two groups (reagent-treated vs. untreated) was validated by a t-test with the same significance level.
APP-D664A inhibits tau hyperphosphorylation at Thr212 and Ser262
The APP-D664A mutation efficiently blocked the truncation of APP at Asp664 when assessed using end-specific APP-α664 antibodies after treatment with 1 μM staurosporine (Fig. 1a). In the absence of additional stimuli, the steady-state cleavage of APP at Asp664 was assessed by coimmunoprecipitation using the anti-GFP and CT-15 antibodies. As the C31 fragment released at position Asp664 of APP (based on the APP695 sequence) is degraded too rapidly to be measured biochemically (Lu et al. 2000), YFP-tagged or GFP-tagged constructs were used. We observed the release of C31–YFP in APPwt–YFP-transfected cells, but not in APP-D664A-YFP-transfected or vector control-transfected cells (Fig. 1b).
Hyperphosphorylated tau is the main component of neurofibrillary tangles. When tau is hyperphosphorylated, it becomes easily detachable and prone to aggregation, forming soluble tau oligomers or filamentous tau inclusions (Hanger et al. 2009), leading to neurotoxicity and synaptic loss. Thus, tau phosphorylation is considered to be an integral factor in AD pathology. To explore the possible relationship of Asp664 cleavage with tau phosphorylation, we compared the level of p-tau at several AD-related epitopes between APPwt-transfected and APP-D664A-transfected cells. Tau phosphorylation at Thr231, Ser199, and Ser396 did not differ depending on APP over-expression or the presence of an Asp664 cleavage site. Tau phosphorylation at Thr212 and Ser262 increased significantly when APPwt was over-expressed. However, when APP-D664A was over-expressed, p-tau phosphorylation was similar to that in the vector control (Fig. 2a). This phenomenon was also observed in primary cultured hippocampal neurons. The immunoreactivity of p-tau at Thr212 and Ser262 was higher in APPwt-transfected cells than in APP-D664A-transfected or vector-transfected cells (Fig. 2b and c). This suggests a relationship between Asp664 cleavage and tau phosphorylation, which is specific to the Thr212 and Ser262 sites.
Blockade of Asp664 cleavage increases PP2A expression and activity
Tau hyperphosphorylation occurs when various kinases and/or phosphatases become dysregulated (Hanger et al. 2009). To identify the kinases or phosphatases that mediate Asp664 cleavage-dependent tau phosphorylation, we evaluated the expression and activation of various kinases and phosphatases involved in tau phosphorylation. There were no significant differences in the expression levels of p-Akt (Ser473), p-GSK-3β (Tyr216), cdk5, p-ERK, or p-PDK1 (Ser241) depending on Asp664 cleavage (Fig. 3a). Next, we explored the expression and activation of PP2A, as it is known to regulate tau phosphorylation in the brain and is related to AD (Liu et al. 2008; Iqbal et al. 2009). The level of the inactive form of PP2A (phosphorylated at Tyr307) was decreased in the APP-D664A-transfected cells, as compared with APPwt (Fig. 3b). Consistent with this result, phosphatase activity was low in APPwt-transfected cells, as compared with APP-D664A, which had activity similar to that in the vector control-transfected cells (Fig. 3c).
Okadaic acid reverses tau dephosphorylation at Thr212 and Ser262 in APP-D664A-transfected cells
To identify the role of PP2A in Asp664 cleavage-dependent tau phosphorylation, N2A cells transfected with individual constructs were treated with okadaic acid (10 nM for 4 h), which is a well-known PP2A inhibitor. The concentration and duration of okadaic acid exposure were chosen based on a previous study in which PP2A activity was inhibited, but apoptosis was not induced in primary cortical neurons (Kickstein et al. 2010). Okadaic acid inhibited PP2A activity efficiently in APP-D664A-transfected and vector-transfected N2A cells (Fig. 3c). In response to okadaic acid, tau phosphorylation at Thr212 and Ser262 increased dramatically in APP-D664A-transfected and vector control-transfected cells, similar to APPwt-transfected cells (Fig. 4). However, tau phosphorylation at Thr231, Ser199, or Ser396 was not significantly changed by okadaic acid treatment. This demonstrates that APP-Asp664 cleavage-dependent tau phosphorylation at Thr212 and Ser262 is mediated by PP2A.
C31 binds PP2A via the NPTY domain and modulates phosphatase activity
The significant effects noted for D664A mutation on tau phosphorylation at Thr212 and Ser262 and on PP2A activity support the role of one or both fragments released from Asp664 cleavage, C31 or Jcasp, in tau phosphorylation. To explore the role of C31 in the regulation of PP2A activity, we transfected N2A cells using the C31–GFP construct or vector control (GFP), and measured PP2A activity. GFP was fused to the C-terminus of C31, to stabilize this short, easily degradable polypeptide. Compared with vector control-transfected cells, PP2A activity was reduced in the C31-transfected cells (Fig. 5a). The level of phosphorylated PP2A, which is the inactive form of PP2A, was increased by C31 over-expression in a dose-dependent manner (Fig. 5b). However, the effect of C31 on PP2A activity and phosphorylation was lower than that of APPwt over-expression. Next, we transfected the NPTY (residues 684–687 of APP695) deletion mutant, C31ΔNPTY, as C31 interacts with adaptor proteins through this region to exert its various functions (reviewed in Zheng and Koo 2006, 2011). We performed coimmunoprecipitation experiments with the anti-PP2A (catalytic subunit) and anti-GFP antibodies, to determine whether there is a direct interaction between these proteins. In the C31-transfected cells, we observed increased binding between PP2A and C31, but not in the C31ΔNPTY-transfected cells (Fig. 5c), which suggests that C31 binds to PP2A through the NPTY region. To determine the effect of this interaction on the regulation of tau phosphorylation, we performed immunoblots using phospho site-specific antibodies. There was a significant decrease in phosphorylation at Thr212 and Ser262 in the C31ΔNPTY-transfected cells (Fig. 5d), as compared with the C31 cells, which was similar to the control vector-transfected cells. This suggests that C31 regulates PP2A activity and tau phosphorylation at Thr212 and Ser262 by interacting with PP2A through the C31-NPTY domain.
Next, we explored the role of the 20-amino acid polypeptide Jcasp, which encompasses the γ- (or ε-) cleavage site and Asp664. Jcasp over-expression had no effect on the activity of PP2A, the level of inactive phospho-PP2A, or the phosphorylation of tau (Fig. 6).
Inhibition of γ-cleavage abrogates the effect of Asp664 cleavage on tau phosphorylation
To explore the relationship between Aβ and Asp664 cleavage-dependent tau hyperphosphorylation, we investigated the levels of tau phosphorylation and PP2A activity in the presence or absence of DAPT treatment in APPwt-transfected and APP-D664A-transfected cells. The inhibition of γ-cleavage of APP with DAPT prevents the release of Aβ from APP, but not the release of C31. Considering that Jcasp exerted negligible effects in our cells in terms of regulating PP2A activity and tau phosphorylation, the blockage of γ-cleavage of APP is useful for exploring the relationships between Aβ and Asp664 cleavage- dependent events. In the APPwt-transfected cells, the phosphorylation of tau at Thr212 and Ser262 was decreased significantly by DAPT treatment (Fig. 7a). However, this inhibitory effect of DAPT was also evident at the Asp664 cleavage-independent tau phosphorylation sites, Thr231, Ser199, and Ser396, and in APP-D664A-transfected and vector control-transfected cells. These results demonstrate that Aβ is a potent inducer of tau phosphorylation, as previously documented (Felice et al. 2008), and that Asp664 cleavage-dependent tau hyperphosphorylation is a downstream process of Aβ. Next, we measured PP2A activity, which was increased in APPwt-transfected cells by DAPT treatment, but not in APP-D664A-transfected and vector control-transfected cells (Fig. 7b). Considering that Aβ increases caspase activation (Gamblin et al. 2003; D'Amelio et al. 2011), it is likely that the effect of Aβ on Asp664 cleavage-dependent events is the result of increased Asp664 cleavage per se (Lu et al. 2003; Shaked et al. 2006). As expected, Asp664 cleavage in APPwt-transfected cells was markedly reduced after treatment with DAPT, as validated in coimmunoprecipitation experiments with anti-GFP and CT-15/6E10 antibodies (Fig. 7c). Taken together, our findings indicate that the mechanism by which DAPT reduces tau phosphorylation at Thr212 and Ser262 is largely attributable to the inhibition of C31 release, which inhibits PP2A activity through a direct interaction.
In this study, we show that Asp664 cleavage increases tau phosphorylation at Thr212 and Ser262 by inhibiting PP2A activity in N2A cells. We also observed a direct interaction between C31 and PP2A mediated by the C31-NPTY region, which was found to play a role in tau phosphorylation. Aβ is intimately connected to this pathway as an upstream component that increases Asp664 cleavage.
The hyperphosphorylation of tau is an integral event leading to AD tau pathology. Among the various tau phosphorylation sites, we analyzed Thr212, Thr231, Ser199, Ser262, and Ser396, as these are known to be associated with AD (Brunden et al. 2009). We observed that tau phosphorylation at Thr212 and Ser262 was decreased in APP-D664A-transfected cells. Cophosphorylation at Thr212, Thr231, and Ser262 plays a key role in inducing caspase activation and neurodegeneration (Alonso et al. 2010). Moreover, Thr212 phosphorylation of tau is known to produce dramatic changes, leading to tau aggregation (Alonso et al. 2010). Thus, the reduction in tau phosphorylation at Thr212 and Ser262 caused by the blockage of Asp664 cleavage may explain, at least in part, how the APP-D664A mutation decreases AD-like pathology (Galvan et al. 2006; Saganich et al. 2006).
After treatment with the PP2A inhibitor okadaic acid, the reduction in tau phosphorylation observed in the APP-D664A-transfected cells was completely abrogated. This suggests that PP2A mediates Asp664 cleavage-dependent tau phosphorylation. Previously, Thr212 and Ser262 were reported as favorable PP2A sites (Kickstein et al. 2010; Qian et al. 2010), which is consistent with our result. PP2A can directly or indirectly regulate tau phosphorylation via the activation of GSK-3β (Qian et al. 2010). In this study, there were no changes in the expression and activation of GSK-3β, as evidenced by immunoblotting for GSF-3β (including the phospho-form). This contradicts a previous report that C31 over-expression increases GSK-3β transcription (Kim et al. 2003). However, C31 was not tagged in the previous study, which meant that it was easily degraded. In addition, the cell line used in that study differed from that used in this study, which might have affected the outcomes. Further investigations are required, including studies of various cells types and in vivo experiments, to resolve this discrepancy and to elucidate the relationship between C31 and GSK-3β.
PP2A is an important phosphatase that mediates tau phosphorylation in AD (Gong et al. 1995; Vogelsberg-Ragaglia et al. 2001; Liu et al. 2008). We demonstrate that PP2A activity decreases with Asp664 cleavage. The over-expression of C31 alone decreased phosphatase activity and increased tau phosphorylation at Thr212 and Ser262. However, we did not identify a significant relationship between Jcasp and PP2A, in contrast to a previous report that Jcasp inhibited PP2A activity by binding to PP2A inhibitor 2 (Madeira et al. 2005). The NPTY region of C31 was shown to be a site for C31 binding to PP2A, which is crucial to the regulation of PP2A activity and tau phosphorylation. Considering that the YENPTY motif is required for the interaction of C31 with various proteins, it is possible that some other binding partner interacts with this PP2A–C31 complex. The effect of C31 over-expression on PP2A activity was smaller than that of APP with Asp664 cleavage site. Considering that the complex of C31 with APP is important to recruit the interacting partners, thereby initiating the signaling pathways (Park et al., 2009), it is possible that the lone over-expression of C31 might be less effective in inhibiting PP2A activity due to less making C31-APP complexes than APP over-expression. To identify the exact reasons of this discrepancy and the mechanism by which C31 regulates PP2A activity after binding, further study is necessary.
Recently, several studies have reported on the critical role of non-apoptotic activation of caspases in the early pathology of AD (de Calignon et al. 2010; Burguillos et al. 2011; Hyman 2011). Caspase cleavage of APP is required for synaptic loss, electrophysiological abnormalities, hippocampal atrophy, and behavioral deficits in AD (Bredesen et al. 2010). Although the in vivo evidence is contradictory (Galvan et al. 2006; Harris et al. 2010), the notion that APP-D664A can reduce the AD phenotype against higher levels of Aβ is generally accepted (Bredesen et al. 2010). In this study, we identified a new mechanism through which the D664A mutation exerts protective effects in AD by decreasing tau phosphorylation at AD-related epitopes and increasing PP2A activity. Considering that Aβ enhances Asp664 cleavage, the modulation of PP2A activity and tau hyperphosphorylation by Asp664 cleavage may be an important mediating factor in Aβ-induced tau pathology.
This study is supported by Korea Research Foundation (KRF) grant funded by the Korea government (MEST, 2009-0067850) and Korean Health Technology R & D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A092004). We are greatly thankful to Professor Chung KC (Yonsei University) for valuable discussion. The authors have no conflict of interest.