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(Circulation Research. 2005;97:144.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Phosphatidylinositol 3-Kinase–Akt Pathway Plays a Critical Role in Early Cardiomyogenesis by Regulating Canonical Wnt Signaling

Atsuhiko T. Naito, Hiroshi Akazawa, Hiroyuki Takano, Tohru Minamino, Toshio Nagai, Hiroyuki Aburatani, Issei Komuro

From the Department of Cardiovascular Science and Medicine (A.T.N., H. Akazawa, H.T., T.M., T.N., I.K.), Chiba University Graduate School of Medicine, Japan; and the Research Center for Advanced Science and Technology (H. Aburatani), University of Tokyo, Japan.

Correspondence to Dr Issei Komuro, Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail komuro-tky{at}umin.ac.jp

	Abstract

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We have recently reported that activation of phosphatidylinositol 3-kinase (PI3K) plays a critical role in the early stage of cardiomyocyte differentiation of P19CL6 cells. We here examined molecular mechanisms of how PI3K is involved in cardiomyocyte differentiation. DNA chip analysis revealed that expression levels of Wnt-3a were markedly increased and that the Wnt/ß-catenin pathway was activated temporally during the early stage of cardiomyocyte differentiation of P19CL6 cells. Activation of the Wnt/ß-catenin pathway during this period was required and sufficient for cardiomyocyte differentiation of P19CL6 cells. Inhibition of the PI3K/Akt pathway suppressed the Wnt/ß-catenin pathway by activation of glycogen synthase kinase-3ß (GSK-3ß) and degradation of ß-catenin. Suppression of cardiomyocyte differentiation by inhibiting the PI3K/Akt pathway was rescued by forced expression of a nonphosphorylated, constitutively active form of ß-catenin. These results suggest that the PI3K pathway regulates cardiomyocyte differentiation through suppressing the GSK-3ß activity and maintaining the Wnt/ß-catenin activity.

Key Words: PI3K • Akt • Wnt • cardiomyocyte differentiation

	Introduction

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The heart is the first organ to be formed from mesodermal cells during embryogenesis, and its developmental process consists of multiple steps such as commitment of immature mesodermal cells into cardiac mesodermal cells, subsequent differentiation of cardiac mesodermal cells into cardiomyocytes, and morphogenesis of the chambered heart. The study of cell fate mapping has revealed that the cells committed into cardiac cell fate are delineated in the posterior lateral region of the epiblast of the mouse embryo during the early gastrulation. These progenitor cells migrate through the node and primitive streak to a cranial destination, and start to express cardiac transcription factors such as Nkx-2.5 and GATA-4.^1,2 After gastrulation, these cells move to the anterior lateral part of the embryo and merge at their anterior margins to form cardiac crescent. Then these cells move ventrally and fuse at the midline to create linear heart tube.³ Advances in molecular biology made it possible to identify many important cardiac transcription factors such as Nkx-2.5, GATA-4, HAND1/2, and MEF2C that serially or synergistically induce the expression of cardiac specific genes and regulate morphogenesis of the developing heart. These genes are also used as a marker for cardiomyocyte differentiation in developing embryos.

Mechanisms of how mesodermal cells are committed to cardiac mesodermal cells, in other words, mechanisms of how expression of these transcription factors are regulated initially, have been largely unknown. Explant cultures of the amphibian and avian embryos revealed that secreted proteins from neighboring endoderm, ectoderm, and the mesoderm itself, play important roles in induction of cardiac transcription factors and differentiation of cardiomyocytes.⁴ Among such molecules, bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF) have been reported to induce cardiomyocyte differentiation from noncardiac mesodermal cells.^5,6 However, several recent studies reported that BMP is required for the maintenance but not for the induction of cardiac transcription factors.^7,8 Moreover, FGF expression seems to be downstream of BMP,⁶ indicating that molecules other than BMP and FGF are required for initial expression of cardiac transcription factors.

Wnt signaling is involved in the development of many organs of various species.⁹ In Drosophila, wingless, a homologue of vertebrate Wnt has been reported to be involved in initial expression of tinman, a Drosophila homologue of Nkx-2.5, through armadillo, a Drosophila homologue of ß-catenin, and drives heart development.¹⁰ In vertebrates, however, canonical Wnt pathway, which uses ß-catenin as a downstream molecule, has been reported to inhibit cardiomyocyte differentiation from cardiac mesoderm.^11–13 On the other hand, Pandur and colleagues reported that Wnt-11, which uses noncanonical Wnt pathway independently of ß-catenin,¹⁴ was required and sufficient for cardiomyocyte differentiation.¹⁵ These reports collectively suggest that noncanonical Wnt pathway plays a positive role, whereas canonical Wnt pathway plays a negative role in cardiomyocyte differentiation.

In spite of the well-designed studies using explant cultures in amphibian or avian embryos, it is difficult to dissect the role of various factors such as BMP, FGF, and Wnt in the cardiac development through the whole process. Moreover, it is hard to elucidate the mechanism of commitment of mesodermal cells into cardiomyocytes in mammalians because of difficulty in explant culture using mammalian embryo of a very early stage. From this viewpoint, in vitro differentiation system, in particular P19CL6 cells, a clonal derivative of murine teratocarcinoma P19 cells, is a very useful model. We have recently demonstrated that TAK-1 and Smad proteins play critical roles in cardiomyocyte differentiation downstream of BMP receptor.^16,17 More recently, Nakamura et al have reported that canonical Wnt pathway is required for differentiation of P19CL6 cells into cardiomyocytes.¹⁸

We have recently reported that phosphatidylinositol 3-kinase (PI3K) pathway is required when mesodermal cells start to express cardiac transcription factors.¹⁹ In this study, we demonstrated that canonical Wnt pathway is required and sufficient for commitment of cardiomyocytes and clarified that PI3K pathway is involved in cardiomyocyte differentiation by crosstalk with canonical Wnt pathway.

	Materials and Methods

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Plasmids and Reagents
Constitutively active Akt (CA-Akt) and dominant negative Akt (DN-Akt) were described previously.²⁰ Mutant ß-catenin (S33A, S37A, T41A, S45A) was from Dr S. Ishihara (Kyoto University, Japan) and Dr T. Noda (The Cancer Institute of the Japanese Foundation for Cancer Research, Japan). pGK-Wnt-3a was from Dr S. Takada (Center for Integrative Bioscience, Okazaki National Research Institutes, Japan). Constitutively active glycogen synthase kinase-3ß (GSK-3ß) was from Dr T. Hagen (Wolfson Digestive Diseases Centre, University Hospital Nottingham, United Kingdom). Constitutively active PI3K (myr-p110) was from Drs M. Kasuga and W. Ogawa (Kobe University, Japan). pTOPFLASH and pFOPFLASH were from Upstate biotechnology. pRL-cytomegalovirus (pRL-CMV) was from Promega. LY294002 was purchased from BioMol Research Laboratories. Akt inhibitor III was purchased from Calbiochem. Recombinant mouse Wnt-3a protein, recombinant-mouse Frizzled-8/Fc chimera protein, and recombinant insulin-like growth factor (IGF) II were purchased from R&D systems.

Cell Culture and Transfections
P19CL6 cells were cultured essentially as described previously.¹⁹ Cells were transfected with plasmids on day 0 of differentiation using the lipofection method as previously described.^16,17 Transfection efficiency was 60% as examined by GFP expression plasmids (data not shown). Luciferase assay was performed essentially as described previously²¹

Immunofluorescence
For evaluating cardiomyocyte differentiation, cells were immunostained using MF20, a monoclonal antibody against sarcomeric myosin heavy chain (MHC). The cells were viewed and photographed with a confocal laser-scanning microscope (Radiance 2000; Bio-Rad). MF20 positive area was calculated and data are expressed as percentage MF20-positive area (pixelxpixel) compared with control P19CL6 cells cultured in differentiation medium.

RT-PCR
RT-PCR was performed as described previously.¹⁹ To confirm that the obtained bands were not derived from contaminated genomic DNA, a negative experiment was done for each sample without reverse transcriptase before PCR (data not shown).

Western Blotting
Total cell lysates, cytosolic, or nuclear fractions were electrophoresed on SDS-polyacrylamide gels. Western blotting was performed as described previously.²²

RNA Preparation and High-Density Oligonucleotide Array Hybridization
Total RNA was extracted from P19CL6 cells on days 0, 4, and 6 of DMSO-induced cardiomyocyte differentiation. Expression profiling was performed using Affymetrix GeneChip MU6500 as described previously.²³

Statistical Analysis
Data are expressed as mean±SE. The significance of differences among means was evaluated using analysis of variance (ANOVA), followed by Fisher PLSD test and Dunnett test for multiple comparisons. Significant differences were defined as P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Akt Acts Downstream of PI3K in Cardiomyocyte Differentiation of P19CL6 Cells
We have recently reported that activation of PI3K is required for DMSO-induced cardiomyocyte differentiation of P19CL6 cells.¹⁹ Akt is one of the important downstream molecules of PI3K. To clarify whether Akt activation is required for cardiomyocyte differentiation, we first examined the effects of a chemical inhibitor of Akt.²⁴ When the Akt inhibitor (10 µmol/L) was added to the medium through day 0 to day 4, the rate of cardiomyocyte differentiation shown by MF20 positive area was markedly decreased, although the Akt inhibitor was less effective than the PI3K inhibitor LY294002 (Figure 1A through 1C). To confirm the contribution of Akt to cardiomyocyte differentiation, we transfected a dominant negative form of Akt into P19CL6 cells. When DN-Akt was introduced into P19CL6 cells, cardiomyocyte differentiation was also suppressed (Figure 1A through 1C). We further investigated whether activation of PI3K/Akt pathway is sufficient to drive cardiomyocyte differentiation of P19CL6 cells. Myristoylated p110, a catalytic subunit of PI3K, leads to constitutive activation of PI3K.²⁵ Direct activation of PI3K by transfecting this construct increased phosphorylation of Akt on Ser-473 to the same degree as DMSO treatment, indicating that PI3K signaling is activated to the same levels in these cells (Figure 1D). However, cardiomyocyte differentiation was not observed (Figure 1E). Moreover, treatment with IGF II (10 ng/mL) also increased phosphorylation of Akt but failed to induce cardiomyocyte differentiation of P19CL6 cells (Figure 1D and 1E). Furthermore, we confirmed that CA-Akt did not induce cardiomyocyte differentiation in the absence of DMSO (Figure 1C). In collection, these results suggest that PI3K/Akt pathway is necessary, but not sufficient for cardiomyocyte differentiation of P19CL6 cells.

View larger version (52K): [in this window] [in a new window]   Figure 1. Akt is required, but not sufficient for cardiomyocyte differentiation. A, Suppression of cardiomyocyte differentiation by inhibiting PI3K/Akt signaling. Cells were cultured in differentiation medium (DM) to induce cardiomyocyte differentiation. On day 0, cells were transfected with DN-Akt or pcDNA3.1. LY294002 (LY) or Akt inhibitor III (Akt-I) were added to the medium for the first 4 days. Cells were stained with MF20 on day 16. Scale bars, 50 µmol/L. B, Quantification of MF20 positive area. At least 4 different fields were measured for each dish. *P<0.001 compared with DM. P<0.05 compared with LY. C, cTnT expression, cells were cultured in DM or in growth medium (GM). On day 0, cells were transfected with DN-Akt, CA-Akt, or pcDNA3.1. Akt inhibitor III (Akt-I) was added to the medium for the first 4 days. D, Phosphorylation of Akt by IGF treatment, cells were cultured in GM or in DM. On day 0, cells were transfected with constitutively active PI3-kinase (myr-p110) or pcDNA3.1. Next day, IGF or LY294002 were added to the growth medium (IGF) or differentiation medium (LY), respectively. Cell lysates were collected 2 days after, and phosphorylation of Akt was examined by Western blotting. E, cTnT expression. IGFII or LY294002 were added from day 0 to day 4 and cTnT expression was examined on day 16 of culture.

Canonical Wnt Pathway Is Activated Transiently Only During the Early Stage of Cardiomyocyte Differentiation
To elucidate the molecular mechanisms of how PI3K/Akt pathway plays a critical role in cardiomyocyte differentiation during the early stage, we searched for the molecules that are activated during this stage, ie, through day 0 to day 4 of P19CL6 cells. By using a gene chip technology, we found that expression of Wnt-3a was increased 12-fold through day 0 to day 4 and decreased 5-fold by day 6 (Table). RT-PCR analysis revealed that Wnt-3a and Wnt-8 but not Wnt-1 were expressed on days 2 and 4, and TOPFLASH activity, which reflects the activity of canonical Wnt signaling, was elevated from day 0 to day 4, consistent with the expression levels of Wnt-3a and Wnt-8 genes (data not shown). These results are in accordance with the previous study by Nakamura et al¹⁸ and indicate that activation of canonical Wnt signaling occurs at the stage when PI3K/Akt signaling is required for cardiomyocyte differentiation, and suggest that canonical Wnt signaling may be a target of PI3K/Akt pathway.

View this table: [in this window] [in a new window]   Table 1. Growth Factors Expressed >3-Fold Through Day 0 to Day 4

Temporal Activation of Canonical Wnt Pathway Is Required and Sufficient for Cardiomyocyte Differentiation
The role of Wnt/ß-catenin pathway, so-called canonical Wnt pathway, in vertebrate cardiomyocyte differentiation and heart development is still controversial.^11–13,18 We first examined the role of canonical Wnt pathway in cardiomyocyte differentiation of P19CL6 cells. To examine whether canonical Wnt pathway is required for cardiomyocyte differentiation, we used extracellular canonical Wnt inhibitor, Fz-8/Fc chimera protein (200 ng/mL), which has been shown to inhibit canonical Wnt signaling.²⁶ When P19CL6 cells were treated with this protein through day 0 to day 4 of differentiation, cardiac troponin T (cTnT) was not expressed and spontaneous beating was not observed (Figure 2A), in accordance with the previous study.¹⁸ Interestingly, when this protein was administered through day 4 to day 8, or day 8 to day 12 of differentiation, cardiomyocyte differentiation was not inhibited (Figure 2A) but spontaneous beating appeared on day 9, a day earlier than without this protein, although there were no molecular evidence, ie, difference in cTnT expression, for the appearance of earlier beating (Figure 2A and data not shown). RT-PCR analysis revealed that administration of this chimera protein from day 0 to day 4 completely abolished expression of Nkx-2.5 and GATA-4 (Figure 2B). These results suggest that activation of canonical Wnt signaling during the early stage is required for DMSO-induced cardiomyocyte differentiation of P19CL6 cells.

View larger version (32K): [in this window] [in a new window]   Figure 2. Temporal activation of canonical Wnt pathway is required and sufficient for cardiomyocyte differentiation of P19CL6 cells. A, Canonical Wnt activity is required for cardiomyocyte differentiation. Cells were cultured in GM or in DM. An extracellular Wnt antagonist, Fz8/Fc chimera protein was added through day 0 to 4, (0–4) 4 to 8 (4–8), and 8 to 12. (8–12). B, Changes in the expression of differentiation markers by inhibition of canonical Wnt pathway. Cells were cultured with (DM+Fz8/Fc) or without (DM) Fz8/Fc protein. RNA was collected on days 0, 2, 4, and 6, and RT-PCR analysis was performed. C, Overexpression of Wnt-3a gene induced cardiomyocyte differentiation without DMSO. Cells were cultured in GM or in DM. On day 0, cells were transfected with pGK-Wnt-3a. D, Administration of soluble Wnt protein in a specific time period (days 0 to 4, 0 to 12). Cells were cultured in growth medium and soluble Wnt-3a protein was added through day 0 to day 4 or through day 0 to day 12.

To examine whether canonical Wnt pathway is sufficient for cardiomyocyte differentiation, we first overexpressed Wnt-3a cDNA in P19CL6 cells and cultured them in growth medium without DMSO. P19CL6 cells expressed cTnT (Figure 2C) and showed spontaneous contraction after 10 days of culture. Next, we cultured P19CL6 cells in growth medium with soluble Wnt-3a protein (100 ng/mL, R&D systems). Surprisingly, when soluble Wnt-3a protein was added to the growth medium continuously, cells never started spontaneous contraction nor expressed cTnT (Figure 2D). However, when the protein was added only during the first 4 days of culture, when canonical Wnt pathway is activated endogenously in DMSO-treated P19CL6 cells, spontaneous contraction was observed from day 10 and cells expressed cTnT (Figure 2D). These results suggest that canonical Wnt signaling has 2 different roles in cardiomyocyte differentiation depending on the differentiation stage of the cells. Although the canonical Wnt pathway promotes commitment into cardiac lineage at the early stage, it inhibits further differentiation into mature cardiomyocytes at the later stage.

PI3K/Akt Pathway Maintains Canonical Wnt Activity Through GSK-3ß During DMSO-Induced Cardiomyocyte Differentiation of P19CL6 Cells
Because canonical Wnt pathway plays an essential role in cardiomyocyte differentiation when PI3K/Akt pathway is required for cardiomyocyte differentiation, we hypothesized that PI3K/Akt pathway is required for the activation of canonical Wnt pathway. To test our hypothesis, we first examined whether CA- or DN-Akt could affect canonical Wnt activity in P19CL6 cells. Consistent with previous studies,^27,28 introduction of CA-Akt or DN-Akt did not change canonical Wnt activity (assessed by TOPFLASH activity) in undifferentiated P19CL6 cells (Figure 3A). Introduction of Akt constructs did not change TOPFLASH activity in L-fibroblasts, NIH3T3 cells, and COS-7 cells (data not shown). However, when P19CL6 cells were treated with DMSO and induced to differentiate into cardiomyocytes, TOPFLASH activity was enhanced by CA-Akt and suppressed by DN-Akt (Figure 3A). These observations indicate that it depends on cell types whether PI3K/Akt pathway activates canonical Wnt pathway or not.

View larger version (33K): [in this window] [in a new window]   Figure 3. PI3K/Akt pathway changes canonical Wnt activity through GSK-3ß in differentiation-induced P19CL6 cells. A, Canonical Wnt activity is altered by PI3K/Akt activity only when cells were treated with DMSO. Cells were cultured in GM or in DM. On day 0, cells were transfected with pTOPFLASH, pRL-CMV, together with or without CA-Akt or DN-Akt. NS, not significant. *P<0.001 compared with DM. B, Phosphorylation of GSK-3ß in P19CL6 cells by inhibition of PI3K/Akt pathway. Cells were cultured in GM or in DM. On day 0, cells were transfected with DN-Akt or pcDNA3.1. Cells were cultured until day 4, and phosphorylation of GSK-3ß was examined by Western blotting. C, Phosphorylation of GSK-3ß by addition of Fz8/Fc chimera protein. D, ß-catenin expression in P19CL6 cells by inhibition of PI3K/Akt pathway. Cells were cultured as described in Figure 3B and fractionated into cytoplasmic and nuclear fraction. Expression of ß-catenin was examined by Western blotting. The same membrane was stripped and labeled with anti-actin (for cytoplasmic fraction) or anti–Topo-1 (nuclear fraction) antibodies to verify even sample loading. D, TOPFLASH activity in P19CL6 cells by inhibition of PI3K/Akt pathway. On day 0, cells were transfected with DN-Akt or pcDNA3.1, pTOPFLASH and pRL-CMV for control. After transfection, medium was changed to GM, DM (DM, DN-Akt), or DM containing LY294002 (LY) or Akt inhibitor III (Akt inhibitor). *P<0.001 compared with GM. P<0.001 compared with DM. E, Expression levels of Wnt-3a and Wnt-8 genes after inhibition of PI3K pathway. Cells were cultured in a DM in the presence (DM+LY) or absence (DM) of PI3K inhibitor, LY294002. RNA was extracted on days 0, 2, 4, and 6, and expression of Wnt-3a and Wnt-8 genes was analyzed by RT-PCR. F, Akt phosphorylation-insensitive mutant GSK-3ß blocked cardiomyocyte differentiation. Cells were cultured in GM or in DM. On day 0, cells were transfected with constitutively-active GSK-3ß (GSK-3ß S9A) or with pcDNA3.1. G, TOPFLASH activity in P19CL6 cells by GSK-3ß S9A. Cells were cultured as described in Figure 4F. On day 0, cells were transfected with GSK-3ß S9A or pcDNA3.1, pTOPFLASH, and pRL-CMV for control. After transfection, medium was changed to GM or DM (DM, GSK-3ß S9A). *P<0.001 compared with DM.

GSK-3ß, a ubiquitously-expressed constitutively-active serine/threonine kinase, regulates a wide variety of cellular functions downstream of signaling pathways including PI3K/Akt pathway and canonical Wnt pathway.²⁹ GSK-3ß activity is suppressed by phosphorylation at the Ser-9 by Akt.³⁰ On the other hand, GSK-3ß phosphorylates and promotes degradation of ß-catenin and downregulates canonical Wnt signaling. By the treatment with DMSO, phosphorylation of GSK-3ß was increased and this increase was suppressed by inhibiting PI3K/Akt pathway both chemically and genetically (Figure 3B). Inhibition of canonical Wnt signaling by Fz-8/Fc protein only slightly decreased GSK-3ß phosphorylation that is increased by DMSO treatment (Figure 3C), whereas it severely decreased cardiomyocyte differentiation. This result suggests that canonical Wnt pathway does not directly interact with PI3K/Akt pathway, and thus we speculate that activation of PI3K/Akt and the following phosphorylation of GSK-3ß by DMSO is not mediated via canonical Wnt signaling. However, further analyses are required to elucidate whether canonical Wnt pathway and PI3K/Akt pathway interact directly or not. Inhibition of PI3K/Akt pathway and phosphorylation of GSK-3ß decreased expression levels of cytoplasmic and nuclear ß-catenin (Figure 3D), and the TOPFLASH activity (Figure 3E) in DMSO treated P19CL6 cells. Inhibition of PI3K/Akt pathway did not alter the expression levels of Wnt-3a and Wnt-8 genes (Figure 3F), indicating that the decrease in the canonical Wnt activity is not attributable to decreased expression levels of Wnt-3a or Wnt-8. To further examine whether decreased cardiomyocyte differentiation by PI3K/Akt pathway inhibition is attributable to decreased Ser-9 phosphorylation of GSK-3ß, we overexpressed GSK-3ß whose Ser-9 is substituted into alanine and insensitive to Akt phosphorylation.³¹ Introduction of this mutant protein into P19CL6 cells on day 0 completely abrogated DMSO-induced cardiomyocyte differentiation and DMSO-induced elevation of TOPFLASH activity (Figure 3G and 3H). These results suggest that when P19CL6 cells are induced to differentiate into cardiomyocytes, PI3K/Akt pathway, activated independently of canonical Wnt signaling, is required to maintain canonical Wnt activity by suppressing GSK-3ß.

PI3K/Akt Pathway and Canonical Wnt Pathway Synergistically Induce Cardiomyocyte Differentiation
If inhibition of PI3K/Akt pathway decreases DMSO-induced cardiomyocyte differentiation in P19CL6 cells by regulating GSK-3ß activity, inhibition of PI3K/Akt pathway should also affect Wnt-3a–induced cardiomyocyte differentiation in P19CL6 cells. To test this, we cultured P19CL6 cells in growth medium with soluble Wnt-3a protein and LY294002 for the first 4 days. Cardiomyocyte differentiation induced by soluble Wnt-3a protein was completely blocked by LY294002 treatment (Figure 4A). We next cotransfected CA-Akt or DN-Akt with pGK-Wnt-3a in P19CL6 cells. As expected, cotransfection of CA-Akt and Wnt-3a elevated TOPFLASH activity and expression levels of cTnT. To the contrary, DN-Akt suppressed Wnt-3a–induced increase in TOPFLASH activity and completely blocked cardiomyocyte differentiation (Figure 4B). Finally, we introduced mutant ß-catenin, which is insensitive to phosphorylation by GSK-3ß, into P19CL6 cells, and examined whether this could rescue cardiomyocyte differentiation of P19CL6 cells. Introduction of the mutant ß-catenin restored cardiomyocyte differentiation, which was suppressed by inhibition of PI3K/Akt pathway (Figure 4C through 4E). These results suggest that PI3K/Akt pathway is required to maintain certain levels of canonical Wnt activity that is enough to induce cardiomyocyte differentiation of P19CL6 cells.

View larger version (49K): [in this window] [in a new window]   Figure 4. PI3K/Akt pathway and canonical Wnt pathway act synergistically to induce cardiomyocyte differentiation in P19CL6 cells. A, LY294002 inhibited soluble Wnt protein-induced cardiomyocyte differentiation. Cells were cultured in GM or in DM. Soluble Wnt-3a protein was added to the growth medium with or without LY294002 through day 0 to day 4. B, Akt activity correlates with canonical Wnt activity in Wnt-3a transfected P19CL6 cells. Cells were cultured in GM. On day 0, cells were transfected with pTOPFLASH, pRL-SV40, pcDNA3.1 (GM), and pGK-Wnt-3a (Wnt-3a) together with or without CA-Akt or DN-Akt. *P<0.001 compared with GM Wnt-3a. C, cTnT expression. Cells were cultured in growth medium. On day 0, cells were transfected with pGK-Wnt-3a (Wnt-3a) together with CA-Akt or DN-Akt. D, Suppression of cardiomyocyte differentiation by inhibiting PI3K/Akt signaling was rescued by activating canonical Wnt pathway. Cells were cultured as described in Figure 1A. On day 0, cells were transfected with DN-Akt or pcDNA3.1, together with or without mutant ß-catenin. Cells were stained with MF20 on day 16. Scale bars, 50 µmol/L. E, Quantification of MF20-positive area. At least 4 different fields were measured for each dish. *P<0.05, P<0.01 compared with mutant ß-catenin nontransfected counterparts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we elucidated the molecular mechanism by which PI3K is critically involved in cardiomyocyte differentiation in vitro. Akt, which was activated by the DMSO treatment, was required for cardiomyocyte differentiation of P19CL6 cells as a downstream of PI3K. Canonical Wnt pathway regulated cardiomyocyte differentiation positively and negatively, depending on the stage of differentiation. Inhibition of PI3K/Akt pathway decreased the content of cytoplasmic and nuclear ß-catenin and the activity of canonical Wnt pathway through decreased GSK-3ß phosphorylation.

Many studies have indicated that PI3K is involved in the differentiation of various kinds of cells such as myoblasts,³² osteoblasts,³³ and adipocytes.³⁴ In this study, Akt was involved in cardiomyocyte differentiation downstream of PI3K. However, introduction of myristoylated, constitutively-active form of Akt could not induce cardiomyocyte differentiation of P19CL6 cells in the absence of DMSO (Figure 1E), suggesting that activation of Akt pathway is required, but not sufficient for cardiomyocyte differentiation.

PI3K/Akt pathway affected cardiomyocyte differentiation through maintaining the activity of the canonical Wnt pathway. Activation of the canonical Wnt pathway was required and sufficient for induction of cardiac transcription factors and cardiomyocyte differentiation in P19CL6 cells. It is noteworthy that this positive regulation of cardiomyocyte differentiation by the canonical Wnt pathway is only temporal, and prolonged activation of canonical Wnt pathway rather inhibited full differentiation into spontaneously contracting cardiomyocytes (Figure 3D). Using Xenopus and chick embryos, it has been reported that inhibitors of canonical Wnt signaling such as crescent and Dkk-1 are secreted from anterior mesoderm and induce cardiogenesis and that ectopic stimulation of canonical Wnt signaling in this area inhibited cardiomyocyte differentiation.^11–13 In those studies, expression of Wnt-3a and Wnt-8, which is transiently observed in all embryonic mesoderm,³⁵ is already limited to posterior mesoderm and is not observed in the anterior mesoderm. The stage when canonical Wnts are expressed corresponds to day 4 or earlier of P19CL6 cell differentiation, indicating that our findings do not contradict with the previous in vivo studies.^11–13 Prolonged exposure to canonical Wnt signaling rather blocked full differentiation into spontaneously contracting cardiomyocyte (Figure 3D). Nakamura et al have reported that canonical Wnt pathway contributes positively to cardiomyocyte differentiation,¹⁸ which is consistent with our results. However, there is some difference between the 2 studies. Activation of the canonical Wnt pathway was sufficient for differentiation into mature spontaneously-contracting cardiomyocytes in our study but not in their study. This discrepancy may come from the duration of Wnt-3a stimulation. We limited the Wnt-3a stimulation to initial 4 days of P19CL6 cell differentiation because thereafter Wnt-3a activation inhibited cardiomyocyte differentiation (Figure 3D). Collectively, our results may explain contradicting results on the requirement of canonical Wnt pathway between in vivo^11–13 and in vitro¹⁸ experiments. Further studies are necessary to elucidate the mechanism of how canonical Wnt pathway shows such biphasic roles in cardiomyocyte differentiation by a differentiation stage-specific manner.

It is still on debate whether canonical Wnt pathway and PI3K/Akt pathway interact with each other through regulation of GSK-3ß.³⁶ Previous reports showed that PI3K/Akt pathway and canonical Wnt pathway is pharmacologically distinct.^27,28 In our study, we observed that PI3K/Akt pathway itself did not activate canonical Wnt pathway but was required for maintenance of canonical Wnt pathway. Our observations do not contradict to previous reports from the point that both signalings are independent and one cannot activate the other pathway. Moreover, Yuan et al²⁷ showed the synergism between PI3K/Akt pathway and canonical Wnt pathway when both signaling pathways are activated, which is consistent with our study (Figures 4A and 5B). In addition, a growing body of evidence suggests that there is a synergistic effect between PI3K/Akt pathway and canonical Wnt pathway in differentiating neuronal cells,³⁷ intestinal cells,³⁸ and myoblasts.³⁹ We speculate that synergistic action between PI3K/Akt pathway and canonical Wnt pathway exists in certain circumstances such as during differentiation of cells or development of organs.

View larger version (19K): [in this window] [in a new window]   Figure 5. Regulation of early cardiomyogenesis by PI3K and the canonical Wnt pathway. After DMSO treatment, undifferentiated P19CL6 cells start to differentiate into cardiac mesodermal cells and mature cardiomyocytes. DMSO treatment activates both the canonical Wnt pathway and the PI3K pathway independently. Activation of the canonical Wnt pathway is required and sufficient for induction of cardiac mesodermal cells. During cardiac mesoderm induction, PI3K pathway enhances activity of the canonical Wnt pathway and both pathways act together in commitment of cardiac mesodermal cells. Once committed into cardiac mesodermal cells, aberrant activation of canonical Wnt signaling rather suppressed further maturation into functional cardiomyocytes.

It should be noted that PI3K/Akt pathway may act as a survival factor for the cardiac mesodermal cells during DMSO- and Wnt-induced cardiomyocyte differentiation in P19CL6 cells in addition to its activity to maintain canonical Wnt activity during cardiomyocyte differentiation.

In summary, we elucidated the molecular mechanisms of how PI3K signaling affects cardiomyocyte differentiation (Figure 5). Canonical Wnt pathway plays a primary and pivotal role in initiation of cardiomyocyte differentiation, and PI3K pathway plays an important role in cardiomyocyte differentiation by maintaining canonical Wnt activity. It is also noteworthy that canonical Wnt pathway is required only temporally and plays both positive and negative roles in cardiomyocyte differentiation by the differentiation stage-dependent manner. Our novel findings in this study could explain contradictory reports between in vitro and in vivo on the role of canonical Wnt pathway in cardiomyocyte differentiation.

	Acknowledgments

 
This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, and Culture; Japan Health Sciences Foundation; Takeda Medical Research Foundation; Takeda Science Foundation; Uehara Memorial Foundation; Kato Memorial Trust for Nambyo Research; Japan Medical Association (to I.K.); Japanese Heart Foundation/Pfizer Japan Grant on Cardiovascular Disease Research; Takeda Science Foundation (to H.A.); Japan Heart Foundation Young Investigator’s Research Grant (to A.T.N.). We thank E. Fujita, A. Furuyama, M. Ikeda, R. Kobayashi, and Y. Ohtsuki for their excellent technical assistance, and Drs S. Ishihara, T. Noda, S. Takada, T. Hagen, W. Ogawa, and M. Kasuga for providing us plasmids.

	Footnotes

 
Original received February 7, 2005; revision received June 16, 2005; accepted June 17, 2005.

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