Wnt Signaling Regulates the Function of MyoD and Myogenin
2000; Elsevier BV; Volume: 275; Issue: 42 Linguagem: Inglês
10.1074/jbc.m004349200
ISSN1083-351X
AutoresAlan G. Ridgeway, Helen Petropoulos, Sharon Wilton, Ilona S. Skerjanc,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe myogenic regulatory factors (MRFs), MyoD and myogenin, can induce myogenesis in a variety of cell lines but not efficiently in monolayer cultures of P19 embryonal carcinoma stem cells. Aggregation of cells expressing MRFs, termed P19[MRF] cells, results in an approximately 30-fold enhancement of myogenesis. Here we examine molecular events occurring during P19 cell aggregation to identify potential mechanisms regulating MRF activity. Although myogenin protein was continually present in the nuclei of >90% of P19[myogenin] cells, only a fraction of these cells differentiated. Consequently, it appears that post-translational regulation controls myogenin activity in a cell lineage-specific manner. A correlation was obtained between the expression of factors involved in somite patterning, including Wnt3a, Wnt5b, BMP-2/4, and Pax3, and the induction of myogenesis. Co-culturing P19[Wnt3a] cells with P19[MRF] cells in monolayer resulted in a 5- to 8-fold increase in myogenesis. Neither BMP-4 nor Pax3 was efficient in enhancing MRF activity in unaggregated P19 cultures. Furthermore, BMP-4 abrogated the enhanced myogenesis induced by Wnt signaling. Consequently, signaling events resulting from Wnt3a expression but not BMP-4 signaling or Pax3 expression, regulate MRF function. Therefore, the P19 cell culture system can be used to study the link between somite patterning events and myogenesis. The myogenic regulatory factors (MRFs), MyoD and myogenin, can induce myogenesis in a variety of cell lines but not efficiently in monolayer cultures of P19 embryonal carcinoma stem cells. Aggregation of cells expressing MRFs, termed P19[MRF] cells, results in an approximately 30-fold enhancement of myogenesis. Here we examine molecular events occurring during P19 cell aggregation to identify potential mechanisms regulating MRF activity. Although myogenin protein was continually present in the nuclei of >90% of P19[myogenin] cells, only a fraction of these cells differentiated. Consequently, it appears that post-translational regulation controls myogenin activity in a cell lineage-specific manner. A correlation was obtained between the expression of factors involved in somite patterning, including Wnt3a, Wnt5b, BMP-2/4, and Pax3, and the induction of myogenesis. Co-culturing P19[Wnt3a] cells with P19[MRF] cells in monolayer resulted in a 5- to 8-fold increase in myogenesis. Neither BMP-4 nor Pax3 was efficient in enhancing MRF activity in unaggregated P19 cultures. Furthermore, BMP-4 abrogated the enhanced myogenesis induced by Wnt signaling. Consequently, signaling events resulting from Wnt3a expression but not BMP-4 signaling or Pax3 expression, regulate MRF function. Therefore, the P19 cell culture system can be used to study the link between somite patterning events and myogenesis. myogenic regulatory factor Sonic Hedgehog signaling molecule Frizzled 1 receptor myocyte enhancer factor 2C lymphoid-enhancer factor 1 T-cell factor bone morphogenic protein kilobase(s) MyHC, myosin heavy chain A family of myogenic basic helix-loop-helix transcription factors (MRFs)1 plays a major role in controlling the events leading to skeletal muscle development (1Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (391) Google Scholar, 2Yun K. Wold B. Curr. Opin. Cell Biol. 1996; 8: 877-889Crossref PubMed Scopus (326) Google Scholar). These transcription factors, MyoD, myf-5, myogenin, and myf-6/MRF-4/herculin (3Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2514) Google Scholar, 4Braun T. Buschhausen-Denker G. Bober E. Tannich E. Arnold H.H. 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In contrast, Wnt7a binds to Fz7 and signals through a β-catenin-independent pathway, utilizing protein kinase C (reviewed in Refs. 21Cossu G. Borello U. EMBO J. 1999; 18: 6867-6872Crossref PubMed Scopus (254) Google Scholar, 35Arias A.M. Brown A.M. Brennan K. Curr. Opin. Genet. Dev. 1999; 9: 447-454Crossref PubMed Scopus (92) Google Scholar, 36Kuhl M. Sheldahl L.C. Park M. Miller J.R. Moon R.T. Trends Genet. 2000; 16: 279-283Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). Both pathways result in the activation of gene expression. Although it is clear that Wnt signaling events result in the expression of MRFs during embryogenesis, a role for Wnt signaling in the regulation of MRF activity has not yet been studied. Another family of signaling molecules involved in patterning the somite are the bone morphogenic proteins (BMPs) (23Tajbakhsh S. Cossu G. Curr. Opin. Genet. Dev. 1997; 7: 634-641Crossref PubMed Scopus (102) Google Scholar, 24Gossler A. 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Pax3, a member of the paired box family of transcription factors, is expressed in the maturing cells of the dorso-medial lip, marking the early stages of myogenic cell specification (40Goulding M. Lumsden A. Paquette A.J. Development. 1994; 120: 957-971PubMed Google Scholar, 41Williams B.A. Ordahl C.P. Development. 1994; 120: 785-796Crossref PubMed Google Scholar). The level of BMPs within the somite, combined with the presence or absence of its antagonist noggin, controls the ability of Pax3-positive cells to activate MyoD and myf-5 expression (42Reshef R. Maroto M. Lassar A.B. Genes Dev. 1998; 12: 290-303Crossref PubMed Scopus (233) Google Scholar). For example, BMP signaling in the absence of noggin inhibits the ability of Pax3 to activate MyoD. Pax3 is necessary for the expression of MyoD in embryos lacking myf-5, indicating that Pax3 functions upstream of MyoD (43Tajbakhsh S. Rocancourt D. Cossu G. Buckingham M. Cell. 1997; 89: 127-138Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). Furthermore, the overexpression of Pax3 in paraxial mesoderm leads to activation of MyoD and myf-5 expression (44Maroto M. Reshef R. Munsterberg A.E. Koester S. Goulding M. Lassar A.B. Cell. 1997; 89: 139-148Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). A tissue culture system capable of emulating early embryonic events that occur during somitogenesis would be valuable for further analysis of the mechanisms involved. The P19 cell culture system may be such a system, because the differentiation of these pluripotent stem cells simulates the biochemical and morphological processes that occur during early embryonic development (45McBurney M.W. Jones-Villeneuve E.M. Edwards M.K. Anderson P.J. Nature. 1982; 299: 165-167Crossref PubMed Scopus (577) Google Scholar, 46Skerjanc I.S. Trends Cardiovasc. 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The appearance of cardiac and skeletal muscle is dependent both on the presence of Me2SO and on unknown factors in the fetal calf serum (50Wilton S. Skerjanc I.S. In Vitro Cell. Dev. Biol. Anim. 1999; 35: 175-177Crossref PubMed Scopus (27) Google Scholar). In co-culture experiments, skeletal muscle development in P19 cells was regulated by factors secreted from the neural tube (51Angello J.C. Stern H.M. Hauschka S.D. Dev. Biol. 1997; 192: 93-98Crossref PubMed Scopus (15) Google Scholar). Thus, P19 cells provide an easily manipulatable system to examine early developmental events in tissue culture. Previous studies examined how the ectopic expression of MyoD affects the developmental potential of P19 cells (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). P19 cells expressing MyoD (termed P19[MyoD] cells), retained stem cell characteristics and did not differentiate into skeletal muscle until the cells were aggregated, either with or without Me2SO. These results suggested that mesoderm induction, via cellular aggregation, was essential for MyoD activity. Similar results were obtained by others in embryonic stem cells (53Shani M. Faerman A. Emerson C.P. Pearson-White S. Dekel I. Magal Y. Symp. Soc. Exp. Biol. 1992; 46: 19-36PubMed Google Scholar). Studies of P19[MyoD] cells have shown that MyoD protein is present and capable of binding DNA both before and after aggregation (54Armour C. Garson K. McBurney M.W. Exp. Cell Res. 1999; 251: 79-91Crossref PubMed Scopus (24) Google Scholar). This finding suggests that cellular aggregation is responsible for initiating signaling cascades that regulate MyoD directly. Alternatively, aggregation may indirectly effect MyoD activity, possibly by inducing the expression of an essential cofactor or by altering chromatin structure at muscle-specific promoters. Previous studies have shown that myogenin, like MyoD, requires cellular aggregation to initiate myogenesis (55Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the present study, we have examined potential mechanisms involved in regulating MRF activity during cellular aggregation. Here we show that myogenin activity appears to be regulated in a cell lineage-specific and post-translational manner. During aggregation, somite-patterning factors such as Wnt3a, BMP-2/4, and Pax3 are expressed. In monolayer cultures, Wnt3a, but not Pax3 or BMP-4, can activate MRF- induced myogenesis in P19 cells, bypassing the requirement for aggregation. All cDNAs in expression vectors are driven by the phosphoglycerate kinase (pgk-1) promoter (56Adra C.N. Boer P.H. McBurney M.W. Gene. 1987; 60: 65-74Crossref PubMed Scopus (308) Google Scholar). The DNA construct PGK-MyoD contains a 1.7-kb EcoRI fragment containing the complete open reading frame of MyoD cDNA (3Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2514) Google Scholar), as described (57Pari G. Jardine K. McBurney M.W. Mol. Cell. Biol. 1991; 11: 4796-4803Crossref PubMed Scopus (73) Google Scholar). The construct PGK-myogenin contains a 1.4-kbEcoRI fragment containing the complete open reading frame of rat myogenin cDNA (7Wright W.E. Sassoon D.A. Lin V.K. Cell. 1989; 56: 607-617Abstract Full Text PDF PubMed Scopus (942) Google Scholar). The construct PGK-Pax3 contains a 2.3-kbEcoRI fragment containing the complete open reading frame of Pax3 cDNA (58Goulding M.D. Chalepakis G. Deutsch U. Erselius J.R. Gruss P. EMBO J. 1991; 10: 1135-1147Crossref PubMed Scopus (754) Google Scholar). The construct PGK-Wnt3a contains a 1.4-kbEcoRI fragment containing the complete open reading frame of Wnt3a (59Roelink H. Nusse R. Genes Dev. 1991; 5: 381-388Crossref PubMed Scopus (240) Google Scholar). The construct PGK-Puro contains the gene encoding puromycin resistance, as described (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). The construct PGK-LacZ contains the gene encoding β-galactosidase. PGK-vector DNA is a plasmid containing the pgk-1 promoter alone. P19 embryonal carcinoma cells were cultured as described (47Rudnicki M.A. McBurney M.W. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells. A Practical Approach. IRL Press, Oxford1987: 19-49Google Scholar, 50Wilton S. Skerjanc I.S. In Vitro Cell. Dev. Biol. Anim. 1999; 35: 175-177Crossref PubMed Scopus (27) Google Scholar) in 5% Cosmic calf serum (Hyclone, Logan, UT) and 5% fetal bovine serum (CanSera, Rexdale, Ontario). Cells were transfected by the calcium phosphate method (60Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4826) Google Scholar) unless otherwise stated. Stable cell lines expressing myogenin, MyoD, or Wnt3a were generated as described previously (55Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 61Ridgeway A.G. Petropoulos H. Siu A. Ball J.K. Skerjanc I.S. FEBS Lett. 1999; 456: 399-402Crossref PubMed Scopus (20) Google Scholar). Duplicate transfections were performed with 8 μg of PGK-myogenin or 8 μg of PGK-MyoD, 1 μg of PGK-Puro, 1 μg of PGK-LacZ, and 2.5 μg of B17 (62McBurney M.W. Fournier S. Schmidt-Kastner P.K. Jardine K. Craig J. Somat. Cell. Mol. Genet. 1994; 20: 529-540Crossref PubMed Scopus (13) Google Scholar). To isolate P19 control cell lines, duplicate transfections were performed with 8 μg of PGK-vector, 1 μg of PGK-Puro, 1 μg of PGK-LacZ, and 2.5 μg of B17. To generate cells expressing both MyoD and myogenin, duplicate transfections were performed with 4.5 μg of PGK-MyoD, 4.5 μg of PGK-myogenin, 1 μg of PGK-Puro, 1 μg of PGK-LacZ, and 2.5 μg of B17. After 24 h, β-galactosidase assays were performed on one set to ensure high transfection efficiency, and 2 × 106 cells were plated in a 150-mm dish and selected for puromycin resistance (2 μg/ml). After 7 days, colonies were isolated for further studies. Cells expressing MyoD, myogenin, and both MyoD and myogenin are termed P19[MyoD] (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar), P19[Mgn] (55Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and P19[MyoD+Mgn], respectively. Differentiation was induced by plating 5 × 105 P19 control, P19[MyoD], P19[Mgn], or P19[MyoD + Mgn] cells into 60-mm bacterial dishes containing either 0.8% Me2SO or no Me2SO. The presence or absence of Me2SO had no effect on the ability of the MRFs to induce skeletal myogenesis. However, only in the presence of Me2SO will control cells differentiate into cardiac muscle on day 5 and skeletal muscle on day 9. Cells were cultured as aggregates for 4 days and then plated in tissue culture dishes and harvested for RNA, protein, or fixed for immunofluorescence, at the time indicated. In the aggregation time course experiment, cells were aggregated for 1–4 days and harvested 1 day after transfer into tissue culture dishes. To determine the effect of Pax3 expression on the activity of MyoD or myogenin, PGK-Pax3 was transiently transfected into 4 P19[Mgn] and 4 P19[MyoD] cell lines. 7 μg of PGK-Pax3 and 1 μg of PGK-LacZ were transfected into P19[Mgn] and P19[MyoD] cells using the FuGene 6 transfection system (Roche Molecular Biochemicals) according to the manufacturer's protocol. After 24 h, cells were plated onto coverslips and allowed to grow in monolayer and fixed on day 6. To produce cells that stably expressed MyoD and Pax3, P19[MyoD] cells were transfected with 10 μg of PGK-Pax3, 1 μg of PGK-Puro, 1 μg of PGK-LacZ, and 2.5 μg of B17, using the CaPO4transfection method (60Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4826) Google Scholar). Transfection efficiencies were confirmed to be high (as above), and clones were selected in puromycin (2 μg/ml) for 10 days. Clones were isolated, and those expressing both MyoD and Pax3 were differentiated as described above. To determine the effects of BMP-4 on the ability of MyoD and myogenin to induce myogenesis, P19[MyoD] and P19[Mgn] cells were grown in monolayer and differentiated (described above) in the presence and absence of 1, 5, 25, 100, and 200 ng/ml BMP-4 (Genetics Institute, Cambridge, MA) and fixed after 2, 4, or 6 days in monolayer culture or after 6 days of differentiation. To determine the effects of Wnt3a on MRF activity, P19[MRF] cells were mixed with P19[Wnt3a] cells in ratios of 1:1 or 1:2 depending on the experiment. A total of 150,000 cells was plated onto gelatin-coated coverslips in 35-mm dishes. Cultures were grown for 6 days before fixing for immunofluorescence. These mixes were also grown in the presence of 5 ng/ml BMP-4 where indicated. Cells were fixed in either methanol at −20 °C for 5 min or Lana's fixative (4% paraformaldehyde, 14% v/v saturated picric acid, 125 mm sodium phosphate) for 30 min, rehydrated in PBS for 30 min at room temperature, and then incubated with the appropriate antibody. For total muscle myosin staining, 50 μl of a mouse anti-MyHC monoclonal antibody supernatant, MF20 (63Bader D. Masaki T. Fischman D.A. J. Cell Biol. 1982; 95: 763-770Crossref PubMed Scopus (796) Google Scholar), was incubated for 1 h at room temperature. For myogenin staining, 100 μl of the anti-myogenin monoclonal antibody supernatant, F5D (64Wright W.E. Dackorytko I.A. Farmer K. Dev. Genet. 1996; 19: 131-138Crossref PubMed Scopus (25) Google Scholar), containing 0.03% Triton X-100 and 5% fetal calf serum, was incubated at 4 °C for 24 h. After three 5-min washes in PBS, cells were incubated for 1 h in 50 μl of PBS with 1 μl of goat anti-mouse IgG(H+L) Cy3-linked antibody (Jackson Immunoresearch Laboratories, PA). Coverslips were mounted in a solution of 50% glycerol, 40% PBS, 9.9% p-phenylenediamine, and 0.1% Hoechst stain. Immunofluorescence was visualized with a Zeiss Axioskop microscope, and images were captured with a Sony 3CCD color video camera, processed using Northern Exposure, Adobe Photoshop, and Corel Draw software, and printed with a dye sublimation phaser 450 Tektronic printer. Immunofluorescence experiments were repeated at least twice with two P19 control cell lines and four P19[Mgn] cell lines. Total RNA was isolated from differentiated P19 control, P19[MyoD], and P19[Mgn] cell cultures by the lithium chloride/urea extraction method before and after differentiation (65Auffray C. Rougeon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2085) Google Scholar). Northern blot analysis was performed as described previously (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). Total RNA (6 μg) was separated on a 1% agarose, formaldehyde gel. Transfer to Hybond-N (Amersham Pharmacia Biotech, Oakville, Canada) occurred by capillary blotting, and RNA was cross-linked by UV irradiation. The membrane was hybridized to DNA probes labeled to over 109 cpm/μg with [α-32P]dCTP using a multiprime labeling kit (Amersham Pharmacia Biotech). The probes were purified on a spin column of Sephadex G-50 (Amersham Pharmacia Biotech) and hybridized for 16 h at 42 °C. Washing was performed for 5 × 5 min at room temperature in 2× SSC, 0.2% SDS and for 15 min at 65 °C in 0.2× SSC, 0.2% SDS (0.1× SSC, 0.2% SDS for Wnt5b blots). Hybridization was visualized by autoradiography and with a PhosphorImager SI from Molecular Dynamics. Densitometry was carried out using ImageQuaNT v1.11 software from Molecular Dynamics. The probes used were: a 600-bp PstI fragment from the human cardiac α-actin last exon (66Rudnicki M.A. Jackowski G. Saggin L. McBurney M.W. Dev. 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Genes Dev. 1991; 5: 381-388Crossref PubMed Scopus (240) Google Scholar), a 440-bp PstI/SmaI fragment of the Wnt5b EST (IMAGE clone ID 439000, obtained from ATCC catalog no. 896114), a 2.2-kb full-length Wnt 1 cDNA (33Tajbakhsh S. Borello U. Vivarelli E. Kelly R. Papkoff J. Duprez D. Buckingham M. Cossu G. Development. 1998; 125: 4155-4162Crossref PubMed Google Scholar), and a 1.6-kb full-length Wnt7a cDNA (33Tajbakhsh S. Borello U. Vivarelli E. Kelly R. Papkoff J. Duprez D. Buckingham M. Cossu G. Development. 1998; 125: 4155-4162Crossref PubMed Google Scholar). The skeletal muscle-specific probe was a 600-bp EcoRI fragment from the rat MLC 1/3 cDNA (68Garfinkel L.I. Periasamy M. Nadal-Ginard B. J. Biol. Chem. 1982; 257: 11078-11086Abstract Full Text PDF PubMed Google Scholar). All blots were standardized using a 750-bpEcoRI fragment of rabbit 18 S cDNA. Previous studies have shown that stable P19 cell lines expressing either MyoD or myogenin required aggregation to initiate myogenesis (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar, 55Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To examine whether the expression of both MyoD and myogenin could bypass the requirement for cellular aggregation, stable cell lines were isolated that expressed both MRFs. Similar to P19[MyoD] cells (52Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar) and P19[Mgn] cells (55Ridgeway A.G. Wilton S. Skerjanc I.S. J. Biol. Chem. 2000; 275: 41-46Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), P19[MyoD+Mgn] cell lines did not express significant levels of MyHC when grown as a monolayer, as indicated by immunoreaction with the anti-MyHC antibody MF20 (data not shown). However, after 4 days of aggregation with (Fig.1, A, C, andE) or without (Fig. 1, B, D, andF) Me2SO, P19[Mgn] (Fig. 1, C andD) and P19[MyoD+Mgn] (Fig. 1, E andF) cells appeared bipolar and expressed MyHC on day 6. P19 control cells did not differentiate into skeletal muscle either with (Fig. 1 A) or without Me2SO (Fig. 1 B) on day 6. P19 control ce
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