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One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.
Polypeptide growth factors, such as insulin, insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang1), exert biological functions through binding to their transmembrane receptors that belong to a large family of receptor tyrosine kinases (RTKs) (4). Consequently, the receptor molecules form homo- or heterodimers, and the intracellular tyrosines at the carboxyl termini of the receptors become phosphorylated (37). Numerous distinct adaptor/regulatory proteins, through their Src homologous 2 (SH2) domains, bind to the phosphotyrosines and transduce the signal to downstream pathways, among which are two essential and well-characterized signaling cascades—the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt signaling pathways (4, 13, 37).
The regulatory subunit of PI3K, p85, possesses the SH2 domain and can, therefore, bind to phosphotyrosines on the RTKs and subsequently render activation of the catalytic subunit of PI3K, p110 (7, 8). Active p110 phosphorylates phosphoinositide biphosphate (PIP2), turning it into PIP3 that recruits PDK1 and Akt to the cellular membrane, where Akt is phosphorylated at threonine 308 (T308 for Akt1) by PDK (5, 23, 30). The serine 473 (S473) of Akt (Akt1) is phosphorylated by mTOR complex 2 (mTORC2) and other kinases (17, 36). Phosphorylation of Akt at these two amino acids brings it to full activation. In PDK1-deficient embryonic stem (ES) cells, T308 phosphorylation was abolished and most of the Akt activity was lost, although the S473 phosphorylation was intact (40).
Akt plays an important role in multiple biological processes, such as cell survival, growth, glucose metabolism, and angiogenesis (2, 12, 14-16, 22, 23, 39, 41-43). In mammals, there are three Akt isoforms, termed Akt 1, -2, and -3. Previously, we generated Akt1- and Akt3-deficient mice and studied their roles in mouse development (2, 15, 39, 42, 43). We found that the Akt1 and -3 double knockout (KO) (DKO) mice were embryonically lethal at around embryonic day 12 (E12) and manifested developmental defects in multiple tissues, including the cardiovascular system (14, 15, 43). These studies suggest that the Akt signaling pathway is involved in cardiovascular development.
Other than Akt isoforms, PDK1 also activates another group of AGC family kinases, such as p70 ribosomal S6 kinase (S6K) (32), serum, and glucocorticoid-induced protein kinase (SGK) (26), p90 ribosomal S6 kinase (RSK) (21), and atypical isoforms of protein kinase C (PKC) (31). Comprehensive and intensive mouse genetic studies performed mainly by Alessi and coworkers have confirmed the regulation of these AGC kinases by PDK1 (3, 9, 10, 27-29, 40).
PDK1 knockout mice were severely growth retarded and died at around E9.0, indicating an essential role of PDK1 in development (27). However, its function and downstream targets in cardiovascular development are still elusive. To study this, we deleted PDK1 specifically in endothelial cells through Cre recombinase-mediated excision (25). The results have revealed an essential role of PDK1 in vascular remodeling and integrity and in cardiac development through activation of Akt and its downstream target of Snail.
Mice were housed in accordance with the regulations on mouse welfare and ethics of Nanjing University in groups with 12-h dark-light cycles and free access to food and water. All procedures were conducted with the approval of the relevant authority. PDK1-floxed mice were provided by Dario Alessi (University of Dundee), and PTEN-floxed mice were as previously described (29, 38). These mice were maintained in B6 genetic background. Primers for genotyping were as follows: PDK1-p99, 5′ ATC CCA AGT TAC TGA GTT GTG TTG GAA G 3′, and PDK1-p100, 5′ TGT GGA CAA ACA GCA ATG AAC ATA CAC GC 3′.
Antibodies for immunofluorescence staining and for Western blotting are as follows: anti-smooth muscle antigen (anti-SMA) (Thermo MS-114-P0), platelet endothelial cell adhesion molecule 1 (PECAM-1)/CD31 (BD Pharmingen 550274), endomucin (eBioscience 14-5851), total Akt (CST 9272), pAKT Thr308 (EPITOMICS 2214-1) and Ser473 (CST 9271), PDK1 (Epitomics 1624-1), S6K (CST 9202), pS6K Ser240/244 (CST 2215), pS6K Thr389 (CST 9205), pS6K Ser235/236 (CST 2211), PRAS40 (CST 2610), pPRAS40 Thr246 (CST 2640), pPKC Thr514 (for pS6K Thr229, CST 9379), pan-actin (Thermo MS-1295-P0), Snail (MN Bioworld BS1853), MF20 (DSHB), fluorescein (fluorescein isothiocyanate [FITC])-conjugated Affinipure goat anti-mouse IgG (Jackson ImmunoResearch 113-095-166), Cy3-conjugated AffiniPure goat anti-rat IgG (Jackson ImmunoResearch 112-165-167), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes), and horseradish peroxidase (HRP)-linked secondary antibodies (Thermos Scientific product no. 31460 and product no. 31430).
Yolk sacs and embryos were collected and snap-frozen in liquid nitrogen until use. Tissue lysates were prepared in lysis buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, 20 mM glycerophosphate, 1% NP-40, 5 mM EDTA, 0.5 mM EGTA, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamidine, 1 mM dithiothreitol [DTT], 50 mM NaF, 4 μM leupeptin, at pH 8.0). Samples were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with TBST (50 mM Tris, 150 mM NaCl, 0.5 mM Tween 20, pH 7.5) and incubated with primary antibodies overnight. The membranes were then incubated with a secondary anti-rabbit or anti-mouse HRP-conjugated antibody, and the signals were detected with enhanced chemiluminescence (ECL). Membranes were blotted for pan-actin for equal loading control after membrane stripping.
The hematoxylin-eosin (HE) and immunofluorescence (IF) protocols were as described previously (42). Briefly, E9.5 to -11 embryos or yolk sacs were first washed with cold phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (PFA) 1 or 2 h at 4°C. The samples were processed successively by (i) a 30-min wash in PBS at 4°C; (ii) a 15-min incubation each in 30%, 50%, 75%, and 85% ethanol and then 2 × 10-min incubation in 95% and 100% ethanol at room temperature (RT); (iii) 3 × 10-min incubation in xylene at RT; (iv) 20-min incubation in paraffin/xylene (1:1) at 65°C; and (v) 3 × 30-min incubation in fresh paraffin at 65°C. The processed samples were then embedded in paraffin and sectioned (6 μm thick), and the sections were stained with HE following standard protocol. For whole-mount IF staining, embryos and yolk sacs were fixed as described above in 4% PFA at 4°C for 1 h and then rinsed twice with PBS. The samples were blocked overnight at 4°C in 3% goat serum (Boster; AR0009). IF staining was conducted using anti-PECAM (CD31) antibody or anti-SMA antibody at 4°C overnight. The samples were washed three times with PBS and incubated with Cy3-conjugated AffiniPure goat anti-rat IgG or FITC-conjugated AffiniPure goat anti-mouse IgG. Fluorescence microscopy images were obtained with a research fluorescence microscope (Olympus) equipped with a digital camera. Images were collected and recorded using Adobe Photoshop 5.0 on an IBM R52 computer. For atrioventricular canal (AVC) ex vivo explant culture IF staining, the samples were fixed with 4% PFA, rinsed with PBS, permeabilized with 0.5% Triton X-100, pH 7.0, and blocked overnight at 4°C in 3% bovine serum albumin (BSA)/0.5% Tween 20 in PBS. The samples were incubated with anti-SMA antibody (dilution 1:500) for 2 h at 4°C and washed with 0.2% BSA/0.5% Tween 20 in PBS. The slides were then incubated with FITC-conjugated AffiniPure goat anti-mouse IgG (1:500). To-Pro-3 (642/661 Molecular Probes T3605) was applied to the samples to display the nucleus. Images were obtained and recorded as described above. Confocal microscope images were obtained using an inverted microscope (IX10; 1X40 Olympus) equipped with a scanning laser system (Fluo-View; Olympus). En face and Z-plane sections were obtained using FluoView software (Olympus).
Rat tail collagen type I (Sigma) was prepared according to the manufacturer's instructions. The solution was dispensed in 24-well dishes and allowed to solidify inside a 37°C, 5% CO2 incubator. Subsequently, collagen gels were washed several times with media containing M199, 5% fetal bovine serum (FBS), and selenium (ITS) plus antibiotics and drained overnight. AVC tissue was dissected in sterile PBS plus antibiotics from E9.5 embryos. AVC explants were placed on drained collagen gels, with the endocardium facing down and allowed to attach for 12 h at 37°C, 5% CO2. Media (0.5 ml/well) were added, and the cultures were reincubated for up to 3 days.
The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed as previously reported (43). Briefly, the sections were treated with proteinase K (20 μg/ml) and incubated with terminal deoxynucleotidyl transferase (TdT) and biotinylated dUTP.
The t test was used for statistical analysis.
PDK1-floxed mice were as previously reported and were maintained in a C57/B6 genetic background (29). To delete PDK1 specifically in endothelial cells, female PDK1-floxed mice (PDK1F/F) were crossed with male Tie-2 Cre mice to obtain female PDK1F/+ mice and male PDK1F/+:Tie-2 Cre mice (25). Mating between the mice of these two genotypes gives rise to PDK1F/F:Tie-2 Cre mice that have the PDK1 deletion in endothelial cells. Mice were genotyped by PCR, and PDK1 loss was confirmed by Western blotting (Fig. (Fig.11 A and B). The result showed that PDK1 levels were dramatically decreased in endothelial PDK1 deletion mice (Fig. (Fig.1B).1B). We also examined the phosphorylation levels of PDK1 downstream targets, such as Akt T308 and S473 and S6K T229 and T389, as well as Akt substrate PRAS40 and S6K substrate S6 in embryos of the control and of endothelial PDK1 deletion. The results showed that the phosphorylation levels of Akt T308 and S6K T229 (PDK1 phosphorylation sites) were significantly reduced in embryos of PDK1 deletion compared to the control (Fig. (Fig.1C).1C). Although Akt T473 and S6K T389 phosphorylation levels were higher in PDK1-deficient embryos than those in control, the activity of Akt and S6K became lower because the phosphorylation levels of their substrates, PRAS40 and S6K, were reduced in PDK1-deficient embryos compared to the control (Fig. (Fig.1C).1C). Furthermore, we performed immunofluorescence staining to show that the T308 phosphorylation of Akt, a residue phosphorylated by PDK1, was nearly absent in the PDK1 deletion endothelial cells (Fig. (Fig.1D).1D). Collectively, these results indicate that PDK1 has been deleted correctly in endothelial cells, which in turn reduced total PDK1 activity in the whole embryo.
At E9.5, endothelial PDK1 deletion mice were nearly as normal as the controls (Fig. (Fig.22 A and B). At E10.5, the endothelial PDK1 deletion mice were growth retarded and displayed hemorrhage and hydropericardium as well as hearts with abnormal morphology (Fig. 2C to F and C′ to F′). The development of these mice stopped, and the mice died at around E11.5 (Fig. 2G and H). These phenotypes indicate that endothelial deletion of PDK1 affects cardiovascular integrity and function, resulting in embryonic mortality.
At E10.5, big vessels were absent in the yolk sacs of endothelial PDK1 deletion mice (Fig. (Fig.33 A and B). The PDK1 deletion embryo also displayed head hemorrhage (Fig. (Fig.3D).3D). Further analysis of the yolk sac by whole-mount PECAM staining showed that the primary capillary plexus was present in PDK1 deletion mice, but these capillary plexuses failed to fuse into big vessels, indicating a vascular remodeling defect (Fig. 3E and F). Consistently, a similar remodeling abnormality was found in the head of the PDK1 deletion embryo (Fig. (Fig.3G3G and and3H).3H). Compared to the control, the PDK1 deletion embryo showed more but thinner vessel branches (Fig. 3G, H, and K).
We also found defective vasculature in the placental labyrinth of PDK1 deletion mice (Fig. 3I and J). While the capillary vessels/villi could be discerned clearly in the control, they were hardly observed with the endothelial PDK1 deletion placenta (Fig. 3I, J, and L).
Smooth muscle cells are involved in vascular remodeling (1, 11). We therefore analyzed these cells in wild-type and PDK1 deletion mice by immunofluorescence staining. In the wild-type control, the smooth muscle cells (SMA positive) were present mainly along the big vessels, indicating normal smooth muscle investment (Fig. (Fig.3M3M to O). In contrast, the SMA-positive cells were fewer and scattered in PDK1 deletion mice (Fig. 3P to R).
Taken together, these results indicate that the vasculature in both the embryonic and extraembryonic tissues of PDK1 deletion mice has developmental and remodeling abnormalities.
As displayed in Fig. Fig.2,2, endothelial PDK1 deletion mice showed hydropericardium, a sign of cardiac dysfunction. We then investigated the cardiac structure by endomucin and MF20 staining to display endothelium and myocardium/trabeculae. As shown in Fig. Fig.44 A to D, the trabeculae were well connected to myocardium of the ventricular wall and the endothelial cells were tidily lining the trabeculae and myocardium in the control. In contrast, the structural integrity between the endothelium and trabeculae/myocardium was disrupted in the PDK1 deletion heart (Fig. 4E to H). Both the endothelium and the trabeculae became sparse and disordered, and the trabeculae lost connection with the myocardium of the ventricular wall (Fig. 4E to H). In addition, the myocardium of PDK1 deletion mice became thinner than that of the control (Fig. 4I to K). As PDK1 was deleted only in endothelial cells, the reduced thickness of the myocardium could be a secondary effect.
At E10.5, mesenchymal cells were found accumulated along the AVC and the cushion developed normally in the control heart (Fig. (Fig.55 A and C). However, the mesenchymal cells were dramatically decreased, and the cushion was undeveloped in the PDK1 deletion heart (Fig. 5B, D, and G). At E11.5, the valves were established in the control, while they could hardly be discerned in the PDK1 deletion mice (Fig. 5E and F). These results indicated that the developmental process of epithelial-mesenchymal transition (EMT) was impaired in the PDK1 deletion AVC.
To further investigate the EMT process during AVC development, we set up an ex vivo explant AVC culture system (6). In the control, cushion mesenchymal cells migrated normally outside the AVC, while few such cells could be seen in the PDK1 deletion AVC (Fig. (Fig.66 A to P). We also counted the numbers of anti-smooth muscle antigen (anti-SMA)-positive mesenchymal cells of outgrowth in each AVC sample and found a substantial reduction in PDK1 deletion samples compared to the control (Fig. (Fig.6Q).6Q). These results confirmed the defective EMT and valve development in the PDK1 deletion heart and demonstrated that PDK1 is essential for AVC EMT and valve development.
The multiple developmental defects in endothelial PDK1 deletion mice indicated that PDK1 was indispensable for endothelial function. We therefore studied endothelial proliferation and survival of PDK1 deletion mice. A bromodeoxyuridine (BrdU) experiment was performed with control and PDK1 deletion mice, and the results were slightly distinguishable between these two genotypes (data not shown). Next, we carried out a TUNEL assay to assess endothelial apoptosis. As shown in Fig. Fig.77 A to E, while nearly no apoptotic cells were detected in the control, the TUNEL-positive endothelial cells could be found in both the endothelium and AVC of the PDK1 deletion heart (Fig. 7A to E).
During EMT, epithelial cells lose adhesive cell-cell junctions and marker gene expression, such as for E-cadherin and endomucin. Instead, the mesenchymal cells generated from the epithelial cells become motile and express marker genes of SMA and Snail, a transcription factor and master gene for EMT that suppresses expression of adhesive molecules but activates transcription of mesenchymal genes (24). Previously, it was reported that activation of Akt induced Snail expression and EMT in squamous cell carcinoma cell lines (20).We, therefore, examined the levels of Snail in the AVC of control and PDK1-deficient mice. As shown in Fig. Fig.8,8, while Snail was widely observed and localized mainly in the nucleus in the control, its expression was absent from a large number of cells and its localization was changed from nucleus to cytosol (Fig. (Fig.8A8A to F and A′ to F′). It was also reported that Snail can be dually regulated by glycogen synthase kinase 3β (GSK3β) through direct phosphorylation, which promotes Snail degradation and translocation from the nucleus to cytosol, resulting in reduced EMT (45). A variant of Snail (Snail S6A that is constitutively active) that abolishes this phosphorylation significantly promotes EMT (45). GSK3β is a well-established Akt substrate, and phosphorylation of GSK3β by Akt causes its inactivation. In the case of PDK1 deletion, Akt activity is low, which may increase GSK3β activity and Snail phosphorylation, leading to reduced EMT. We therefore delivered adenoviral Snail S6A to the AVC explants and found that, while empty adenovirus (Ad-green fluorescent protein [GFP]) failed to activate EMT, Ad-Snail S6A (constitutively active) effectively rescued the EMT defects in the explants of PDK1 deficiency (Fig. 8G to L).
To investigate whether the EMT defects in the AVC explant of PDK1 deletion mice were due to Akt inactivation, we delivered adenovirus expressing constitutively active Akt to the AVC explant culture. Three out of five AVC cultures showed a complete rescue of EMT defect in PDK1-deficient AVC (Fig. (Fig.99 A to D).
PTEN negatively regulates PDK1-Akt activation (23). Therefore, we next studied the effects of PTEN loss on cardiovascular development of PDK1 deletion mice by crossing PTEN-floxed and PDK1-floxed mice together with Tie-2 Cre mice (PDK1F/F:PTENF/F:Tie2-Cre). Because endothelium-specific deletion of PDK1 resulted in embryonic mortality at around E11.5 (Fig. 2G and H), we dissected the embryos at this stage. Out of four PTENF/F:PDK1F/F:Tie2-Cre mice analyzed, two appeared normal, and histological analysis revealed a level of development of the AVC cushion comparable to that of the control (Fig. 9E to L). At E12.5, we observed six viable embryos out of 18 double knockout mice. Therefore, the loss of PTEN can postpone embryonic lethality by 1 day. Collectively, these data showed that increased Akt activity could rescue the heart phenotype of PDK1-deficient mice, and the loss of PTEN may enhance Akt activity through increased phosphorylation of S473 of Akt in the absence of PDK1.
In this study, we have revealed the crucial function of PDK1 in endothelial cells and cardiovascular development through activation of Akt and Snail. Loss of PDK1 in endothelial cells caused impaired vascular integrity, remodeling, and development and embryonic lethality at around E11.5. In the heart, endothelial deletion of PDK1 affected endocardiac development, including EMT in the AVC and valve formation. As a result of poor development of the endothelium, the structure of the trabeculae and myocardium was disrupted. Thus, our study has demonstrated that PDK1 plays an indispensable role in the development of the cardiovascular system.
VEGF is essential for endothelial proliferation, survival, and migration (1, 11). After binding to its receptor of VEGFR, VEGF activates two essential intracellular signaling pathways of MAPK and PI3K-PDK1-Akt. Similarly, both angiopoietin 1 (Ang1) and its receptor, Tie2, are required for vascular remodeling and vessel integrity. Recently, it was reported that Akt is the major downstream effector of Ang1-Tie2 signaling (18, 35). Therefore, the developmental and functional defects in the absence of PDK1 in endothelial cells could be a consequence resulting from the disruption of VEGF and Ang1 signaling. This is consistent with a previous report using PDK1 KO ES cells to study cell migration (34). It was shown that PDK1 plays an essential role in regulating endothelial cell migration in response to VEGF-A stimulation (34).
Recently, it was shown that PI3K KO mice displayed defects in angiogenesis and vascular integrity (19, 44). In these mice, the activation of Akt was impaired, which is the possible cause for vascular abnormalities (19, 44). Previously, we studied the Akt1 and Akt3 double knockout (DKO) mice (41, 43). These mice were embryonically lethal at around E12 and displayed developmental defects in multiple organs and tissues, including the cardiovascular system (43). Endothelium-specific PDK1 deletion in mice also caused embryonic mortality at around E11.5 and cardiovascular defects. These similarities between the endothelial PDK1 deletion and Akt1 and -3 DKO mice suggest that, first, Akt proteins are the major downstream players of PI3K-PDK1 signaling among the AGC kinases in the endothelium and, second, the phenotype in Akt 1 and -3 DKO mice could be attributed to disrupted endothelial development and function. Data presented here support this idea because delivery of active Akt to the AVC explant of PDK1-deficient mice rescued EMT defects, and enhancement of Akt activation by PTEN deletion resulted in normal AVC development and survival of PDK1-deficient mice at approximately E12. In the future, it will be of great interest to study whether overexpression of Akt could rescue the phenotype of endothelial PDK1 deletion mice. However, this might be a big challenge as transgenic mice with Akt overexpression in endothelial cells were embryonically lethal and showed excessive aberrant vasculature (33). On the other hand, this suggests that balanced and refined regulation of PDK1-Akt signaling is crucial for cardiovascular development, as either a higher intensity (in the case of Akt transgenic mice) or less activity (in the case of PDK1 and Akt KO mice) of Akt signaling gives rise to developmental abnormalities.
In this study, we have demonstrated that deletion of PDK1 in endothelial cells had nearly no impact on cell proliferation. Instead, we found that PDK1 played a role in promoting cell survival, as loss of PDK1 caused endothelial apoptosis. One of the important functions of Akt is antiapoptosis and prosurvival (12). Therefore, the increased endothelial cell death might be due to inactivation of Akt.
Our study was consistent with a previous report showing that Akt activation promoted Snail expression (20). We found that Snail was ubiquitously expressed in the AVC of the control but was absent from a large amount of cells in the PDK1 deletion AVC. In addition, its cellular localization was changed from the nucleus in the control to the cytosol in PDK1 deletion cells. As Snail is a transcription factor and a master gene for EMT that activates mesenchymal gene expression while suppressing the transcription of adhesive molecules of epithelial cells, the changes of Snail expression patterns may reflect the deficit of EMT in the PDK1 deletion AVC. GSK3β was reported to phosphorylate Snail, resulting in Snail translocation to the cytosol and degradation (45). Because GSK3β has been the best-characterized substrate of the Akt kinase and the phosphorylation of GSK3β by Akt suppresses GSK3β activity, the distribution and cellular localization of Snail in PDK1 deletion cells could be due to loss of activity of PDK1-Akt signaling. Our results have demonstrated that Snail S6A (constitutively active) rescues EMT defects in PDK1 deficiency and confirmed this hypothesis.
Another intriguing finding of this study is that the loss of PTEN can rescue the phenotype of PDK1 deficiency. Mechanistically, PTEN loss in the absence of PDK1 may compensate Akt activity through enhancement of Akt S473 phosphorylation. We found a dramatic increase of Akt S473 phosphorylation in cardiomyocyte-specific PTEN/PDK1 double deletion mice (data not shown). Hence, our study suggests that Akt S473 phosphorylation might be PI3K dependent.
The data presented here could be promising for therapeutic applications. In the future, it will be helpful to modulate Akt signaling to improve cardiovascular complications in human patients.
We are grateful to Dario Alessi at the University of Dundee, United Kingdom, for providing the PDK1-floxed mice and Xiao Yang in Beijing for providing the PTEN-floxed mice.
This work was supported by the National Key Basic Research Program of China (2006CB943503) and the National Science Foundation of China (30500264, 30671040, and 30770893) with grants to Zhongzhou Yang and Xinli Li.
Published ahead of print on 10 May 2010.