14-3-3σ Regulates β-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3β

Background Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. Methodology and Principal Findings In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding. Significance Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.


Introduction
Embryonic stem (ES) cells are pluripotent cells that possess selfrenewal properties and retain the capacity for differentiation into all 3 germ layer cells [1,2]. Because of their high proliferation capability, pluripotency and low immunogenicity, ES cells are considered to be a valuable source for cell therapy, tissue regeneration, drug testing and developmental biology [3,4]. ES cell proliferation and renewal are maintained by diverse factors that activate the renewal genetic program via selective signaling pathways [5,6] among which the b-catenin pathway plays a pivotal role [7,8]. At the basal state, b-catenin is associated with a multiprotein destruction complex composed of APC (adenomatous polyposis coli), axin, casein kinase 2 and glycogen synthase kinase 3b (GSK-3b) where it is phosphorylated and degraded via ubiquitin/proteasome [9][10][11]. Upon Wnt activation through binding to frizzled and/or LRP5/6 receptors, disheveled (Dvl) displaces GSK-3b from the APC complex resulting in reduced bcatenin degradation, and increased cytosolic b-catenin which is translocated to nucleus where it is associated with Tcf/Lef transcription factor to drive the expression of renewal and proliferative genes. Experimental data have provided convincing evidence for the crucial role of GSK-3b/b-catenin in ES cell renewal [12][13][14]. GSK-3b is a serine/threonine protein kinase which was originally discovered as an enzyme that phosphorylates and inactivates glycogen synthase in response to insulin, and was subsequently reported to phosphorylate b-catenin and facilitate bcatenin ubiquitination and degradation [15]. GSK-3b inhibition was shown to maintain ES cells in the renewal state [14]. Thus, GSK-3b occupies a central position in controlling b-catenin and ES cell renewal and differentiation. Its activity must be tightly regulated. However, little is known about its regulation in ES cells. We propose in this study that 14-3-3 proteins regulate GSK-3b availability.

Materials and Methods
Cell Culture and Reagents CCE, a mESC line derived from the 129/Sv mouse strain, was obtained from StemCell Technologies with permission from Drs. Robertson and Keller (Vancouver, Canada). CCE cells were cultured on gelatin-coated dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 10 ng/mL leukemia inhibitory factor at 37uC in a humidified 5% CO 2 atmosphere [23,24]. D3 and R1 mouse ES cells [25,26] were cultured and maintained on feeder cells comprising mitotically inactivated primary mouse embryonic fibroblasts (MEFs) in the same medium of CCE cells. Mouse recombinant Wnt3a and Wnt inhibitor, DKK-1 were from Calbiochem (San Diego, CA, USA). The PI3-K inhibitor wortmannin was from Sigma (St. Louis, MO, USA).

Preparation of Nuclear Proteins
Nuclear proteins were extracted by using an extraction kit (Chemicon). Briefly, cells were harvested and lysed with cytoplasmic buffer containing protease inhibitors for 15 min at 4uC, mixed, and centrifuged at 8,000 g for 20 min at 4uC. Supernatants were collected (cytoplasmic extraction) and pellets were resuspended in nucleus buffer containing protease inhibitors for 15 min at 4uC. The resuspended sample was mixed and centrifuged at 16,000 g for 10 min at 4uC. The supernatant containing nuclear extraction proteins was collected and stored at 280uC.

Cell Proliferation Analysis
For cell proliferation analysis in this study, 1.5610 5 transfected cells were seeded (defined as 0 h) and incubated for the indicated time. Cell number was determined by trypan blue assay. Cells were trypsinized, resuspended in medium, and viable cells were counted by using a hemocytometer. Cell proliferation was analyzed with a bromodeoxyuridine (BrdU) cell proliferation assay kit (Chemicon). Briefly, BrdU, a thymidine analog, is incorporated into newly synthesized DNA as cells enter the S phase. Following partial denaturation of double-stranded DNA, BrdU was detected immunochemically with a specific mouse monoclonal antibody. The amount of BrdU was determined after the addition of IgG-peroxidase conjugated secondary antibody, peroxidase substrate and stop solution.
Promoter Activity Assay b-catenin promoter activity was measured by using TOP-FLASH/FOPFLASH reporter (Millipore). TOPFLASH/FOP-FLASH constructs and 14-3-3s or control vectors were incubated with Effectene transfection reagent in a 12-well plate for 48 h. Cells were washed with PBS and lysed in lysis buffer (Promega). Luciferase activity was measured with Luciferase Assay Reagent (Promega), and the emitted light was determined in a luminometer.

Immunoprecipitation and Ubiquitination Assay
CCE cells were transfected with 14-3-3s-Flag or co-transfected with b-catenin and/or GSK-3b for 48 h. Cells were harvested and lysed in RIPA buffer for 30 min at 4uC. After centrifugation, cell lysates were immunoprecipitated with a mouse monoclonal anti-Flag, anti-HA antibodies or mouse IgG as a control. The immunoprecipitates were resuspended in Laemmli sample buffer with 2-mercaptoethanol and boiled for 15 min. Proteins in the immunoprecipitate were separated by SDS-PAGE and analyzed by immunoblotting with rabbit polyclonal antibodies against bcatenin, GSK-3b, axin or APC. To evaluate the isoform-specific interaction of 14-3-3 with GSK-3b, each Flag-tagged 14-3-3 isoform expression vector was transfected into CCE cells, immunoprecipitated with an anti-Flag antibody, then immunoblotted with anti-GSK-3b antibody. To investigate the phosphorylated residue of GSK-3b that is involved in interaction with 14-3-3s, GSK-3b wild-type (WT), GSK-3b S9A mutant or GSK-3b T309A mutant constructs were co-transfected with 14-3-3s-Flag vector. Transfected CCE cells were immunoprecipitated with anti-Flag antibody, then immunoblotted with anti-GSK-3b antibody.
For assay of b-catenin ubiquitination, CCE cells were cotransfected with 14-3-3s-Flag or HA-b-catenin expression vectors for 46 h, then incubated with MG-132 (10 mM) for an additional 2 h. Cell lysates were harvested, immunoprecipitated with a mouse monoclonal anti-HA antibody, and immunoblotted with rabbit polyclonal antibodies against ubiquitin (Cell Signaling).

GSK-3b Activity Assay
Assay of GSK-3b activity was based on measuring tau phosphorylation at Ser-396 and Ser-199 [36,37] by using an ELISA kit (Invitrogen). In brief, CCE cells were co-transfected for 48 h with GSK-3b expression vector and 14-3-3s-Flag vectors or their respective control vectors. The transfected CCE cells were lysed with RIPA buffer and immunoprecipitated with a specific antibody against GSK-3b. The immunoprecipitate was washed and incubated with an assay buffer containing 100 mM ATP and recombinant Tau proteins, then with an antibody against phospho-Ser396 of Tau, a secondary antibody, and substrates. The reaction was terminated by adding a stop reagent, and the optical density of the sample was analyzed at 450 nm in an ELISA reader. Values of Tau phosphor-Ser396 were normalized to the total protein level of Tau.

Statistical Analysis
Differences between groups were analyzed by Student t test. A p value less than 0.05 was considered statistically significant.

14-3-3s Promotes mESC Proliferation via b-catenin
To determine whether b-catenin is involved in 14-3-3smediated mESC proliferation, CCE cells were co-transfected with 14-3-3s overexpression vector and b-catenin siRNA. Suppression of b-catenin expression in CCE transfected with a previously described siRNA [30,31] (Figure 3A) resulted in abrogation of 14-3-3s-induced cell number increases ( Figure 3B). These results were confirmed by another siRNA sequence of b-catenin which exhibited a lesser effect on suppressing b-catenin and a correlated lesser effect on reducing CCE proliferation ( Figure S3). To investigate the relationship between 14-3-3s and b-catenin, we transfected CCE cells with 14-3-3s and analyzed b-catenin phosphorylation, ubiquitination, degradation and nuclear translocation. Phosphorylation and ubiquitination of b-catenin were significantly decreased ( Figure 4A and 4B), whereas total b-catenin protein level was increased ( Figure 4C, upper panel) in 14-3-3s overexpressed CCE cells. Furthermore, b-catenin protein levels were increased in the nuclear fraction ( Figure 4C, lower panel). Thus, 14-3-3s prevents b-catenin from phosphorylation, ubiquitination and degradation, thereby increasing b-catenin protein stability and nuclear translocation.
To ensure that nuclear b-catenin is active in promoting bcatenin/Tcf-targeted gene expression in mESCs, we analyzed bcatenin transcriptional activity by using TOPFLASH/FOP-FLASH reporters. 14-3-3s overexpression increased the promoter activity by ,5-fold over the control ( Figure 4D). We next determined the expression of cyclin D1, one of the b-catenin target genes that are critical in cell cycle progression and cell proliferation. Cyclin D1 protein level was increased by ,3-fold in 14-3-3s-overexpressed cells ( Figure 4E). Taken together, the results indicate that 14-3-3s promotes mESC proliferation via bcatenin.

Discussion
Our findings provide important information regarding the novel role of 14-3-3s in regulating mESC proliferation. Despite the expression of all seven isoforms of 14-3-3 proteins in mESCs, only 14-3-3s participates in mESC proliferation by binding, sequestration and inactivating GSK-3b. Our results demonstrate that 14-3-3s overexpression enhances GSK-3b phosphorylation and inactivation as well as increases interaction between 14-3-3s and GSK-3b. Furthermore, 14-3-3s overexpression triggers dissociation of b-catenin from the APC/ axin/GSK-3b complex, the so-called multiprotein destruction complex. Since the transcriptional bioavailability of b-catenin is tightly controlled by GSK-3b in the destruction complex, our data lead us to conclude that 14-3-3s is capable of sequestering GSK-3b and thereby releasing b-catenin from the multiprotein destruction complex which translocates into the nucleus and carries out the proliferative transcription.
GSK-3b inactivation depends on phosphorylation within the multiprotein destruction complex. At resting state, GSK-3b is active in phosphorylating b-catenin to induce its degradation via ubiquitination/proteasome. When stimulated by Wnt, GSK-3b is phosphorylated and dissociated from the multiprotein destruction complex, thus releasing b-catenin. It is generally thought that phosphorylated GSK-3b is rapidly dephosphorylated and reassociated with the APC/axin complex. In this study, we provide evidence that phosphorylated GSK-3b is controlled by 14-3-3s. High levels of 14-3-3s sequester and inactivate GSK-3b via which they enhance Wnt signaling to increase b-catenin. It is interesting that DKK blocks the effect of Wnt3a as expected but did not interfere with action of 14-3-3s on GSK-3b binding. These findings indicate that 14-3-3s provides a discrete pathway to control GSK-3b availability and activity.
It is well recognized that RA induces ES cells to undergo differentiation and proliferation arrest. A number of mechanisms of RA actions have been proposed but the exact mechanisms are not clear. We show in this study that 14-3-3s/GSK-3b pathway is involved in RA-induced inhibition of mESC proliferation. RA selectively suppresses 14-3-3s. It increases b-catenin phosphorylation and reduces b-catenin resulting in reduction of mESC proliferation. High levels of 14-3-3s confer resistance to RA by restoring GSK-3b phosphorylation and sequestration. Thus, 14-3-3s is pivotal in regulating GSK-3b/b-catenin bioavailability as illustrated in Fig. 8.
Our results reveal that knockdown of 14-3-3s with siRNA reduces mESC proliferation by only 30-40% compared to control ( Figure 1C and 1D), suggesting that mESC proliferation does not depend entirely on 14-3-3s. A compensatory effect may be regulated by other signal pathways. This notion was supported by a recent report which indicates that 14-3-3s-deleted mESC give rise to viable mice with B-cell developmental defects [40]. It is interesting that of all seven 14-3-3 isoforms expressed in mESCs, only 14-3-3s is involved in regulating b-catenin-mediated mESC proliferation. In contrast, 14-3-3f was reported to bind GSK-3b and enhances Tau phosphorylation in brain [19], and 14-3-3f was reported to facilitate b-catenin export from the nucleus and thereby reduces b-catenin transcriptional activity [22]. Reasons for differential regulation of GSK-3b and b-catenin by different 14-3-3 isoforms in different tissues and cells are unclear and require further investigation.
It is interesting to note that b-catenin siRNA does not completely abrogate the enhancing action of 14-3-3s on cell proliferation ( Figure 3B). It is possible that 14-3-3s may enhance cell proliferation by multiple mechanisms. Besides the Wnt/bcatenin mechanism, 14-3-3s may bind phosphorylated Raf-1, activate Raf-1 and its downstream signaling pathway [41,42]. Moreover, 14-3-3 was found to regulate the mammalian target of rapamycin (mTOR) pathway by interacting with tuberous sclerosis complex 2 (TSC2), and sequestering it from binding to mTOR complex, thereby increasing the mTOR activity on de novo protein synthesis and cell proliferation [43,44].
In summary, this study shows for the first time that 14-3-3s regulates mESC proliferation by binding and sequestering GSK-3b as well as inducing GSK-3b phosphorylation and inactivation in a PI-3K/Akt-dependent manner. 14-3-3s is a novel target for ES cell expansion.