A Wnt- and beta -catenin-dependent pathway for mammalian cardiac myogenesis - PubMed (original) (raw)

A Wnt- and beta -catenin-dependent pathway for mammalian cardiac myogenesis

Teruya Nakamura et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Acquisition of a cardiac fate by embryonic mesodermal cells is a fundamental step in heart formation. Heart development in frogs and avians requires positive signals from adjacent endoderm, including bone morphogenic proteins, and is antagonized by a second secreted signal, Wnt proteins, from neural tube. By contrast, mechanisms of mesodermal commitment to create heart muscle in mammals are largely unknown. In addition, Wnt-dependent signals can involve either a canonical beta-catenin pathway or other, alternative mediators. Here, we tested the involvement of Wnts and beta-catenin in mammalian cardiac myogenesis by using a pluripotent mouse cell line (P19CL6) that recapitulates early steps for cardiac specification. In this system, early and late cardiac genes are up-regulated by 1% DMSO, and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were induced days before even the earliest cardiogenic transcription factors. DMSO induced biochemical mediators of Wnt signaling (decreased phosphorylation and increased levels of beta-catenin), which were suppressed by Frizzled-8Fc, a soluble Wnt antagonist. DMSO provoked T cell factor-dependent transcriptional activity; thus, induction of Wnt proteins by DMSO was functionally coupled. Frizzled-8Fc inhibited the induction of cardiogenic transcription factors, cardiogenic growth factors, and sarcomeric myosin heavy chains. Likewise, differentiation was blocked by constitutively active glycogen synthase kinase 3beta, an intracellular inhibitor of the Wntbeta-catenin pathway. Conversely, lithium chloride, which inhibits glycogen synthase kinase 3beta, and Wnt3A-conditioned medium up-regulated early cardiac markers and the proportion of differentiated cells. Thus, Wntbeta-catenin signaling is activated at the inception of mammalian cardiac myogenesis and is indispensable for cardiac differentiation, at least in this pluripotent model system.

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Figures

Figure 1

Figure 1

Wnt3A and Wnt8 are early responses in differentiating P19CL6 cells. (A) Gene expression was determined by QRT-PCR, normalized to GAPDH, and represented as relative copy number. (B) Western blot, showing induction of GATA4 and sarcomeric MHCs vs. tubulin as the control. (C) Immunocytochemistry, showing induction of sarcomeric MHCs. Cells were cultivated for 12 days with (Right) or without (Left) 1% DMSO, and stained with MF20 (red) and 4′,6-diamidino-2-phenylindole (blue). (Bar = 50 μm.) (D) FGF8 expression by RT-PCR.

Figure 2

Figure 2

The Wnt/β-catenin-signaling pathway is activated at the onset of cardiac myogenesis. (A) Increased soluble β-catenin. Cells were treated with (+) or without (−) 1% DMSO for 3 days. The cytosolic fraction was subjected to Western blot analysis. β-Catenin was specifically decreased by Fz-8/Fc, an extracellular antagonist of Wnt signaling. (B) Decreased phosphorylated β-catenin and increased total β-catenin in whole-cell lysates. Cells were cultured as in A and Western blotting was done by using Abs to phospho-β-catenin (Top) and total β-catenin (Middle). (C) Decreased phosphorylated β-catenin (green), shown by immunostaining. (Left) Without DMSO. (Right) With DMSO. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). (Bar = 50 μm.) (D) TCF/lymphoid enhancer factor-dependent transcription. Cells were cultured ± DMSO and transfected with TOPFLASH or FOPFLASH (inactive, mutant TCF sites) along with pRL-CMV. Luciferase activity was determined after 4 days of treatment.

Figure 3

Figure 3

The Wnt/β-catenin-signaling pathway was required for cardiac differentiation. (A) Fz-8/Fc suppresses the induction of GATA4, TBX5, BMP2, BMP4, FGF8, and α-MHC, determined by QRT-PCR. Cells were treated with DMSO plus diluent or 200 ng/ml Fz-8/Fc. Equivalent results were obtained by using Fz-4/Fc. (B Left) Fz-8/Fc suppresses the induction of sarcomeric MHC protein. (Right) Constitutively active GSK-3β (GSK3βA9) suppresses the induction of sarcomeric MHC protein. (Bar = 25 μm.) (C) GSK3βA9 inhibits Wnt3A-induced TCF transcriptional activity. 293T cells were cotransfected with PGK-neo, PGK-Wnt3A, and pcDNA3-GSK-3βA9-HA as shown, plus the TOPFLASH and pRL-CMV reporter genes. (D) GSK-3βA9-HA expression in stably transformed P19CL6 cells, by RT-PCR. Cells were stably transfected with the pcDNA3 expression vectors shown. Plasmid, 5 ng of pcDNA3-GSK-3βA9-HA as template. (E) GSK-3βA9 suppresses DMSO-induced cardiac gene expression.

Figure 4

Figure 4

The Wnt/β-catenin-signaling pathway enhances cardiac myogenesis. (A) Wnt3A CM increases TCF-dependent transcription. P19CL6 cells cotransfected with TOPFLASH and pRL-CMV were assayed after 18 h in Wnt3A or control (neo) CM. (B) Wnt3A CM induces cardiac-specific markers (0–15 days) and BMPs (6 days), shown by QRT-PCR. Wnt3A CM induces sarcomeric MHC, shown by immunostaining. (Bar = 50 μm.) (C) LiCl enhances cardiomyogenesis. Induction of cardiac-specific markers was analyzed by QRT-PCR (day 5). NaCl was added as the control.

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