Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative β-catenin

<p>Abstract</p> <p>Background</p> <p>Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation.</p> <p>Results</p> <p>Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a β-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative β-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4.</p> <p>Conclusion</p> <p>RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating β-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.</p

Recent advances in large-scale protein interactome mapping [version 1; referees: 3 approved]

Protein-protein interactions (PPIs) underlie most, if not all, cellular functions. The comprehensive mapping of these complex networks of stable and transient associations thus remains a key goal, both for systems biology-based initiatives (where it can be combined with other ‘omics’ data to gain a better understanding of functional pathways and networks) and for focused biological studies. Despite the significant challenges of such an undertaking, major strides have been made over the past few years. They include improvements in the computation prediction of PPIs and the literature curation of low-throughput studies of specific protein complexes, but also an increase in the deposition of high-quality data from non-biased high-throughput experimental PPI mapping strategies into publicly available databases

β-catenin is essential for efficient in vitro premyogenic mesoderm formation but can be partially compensated by retinoic acid signalling.

Previous studies have shown that P19 cells expressing a dominant negative β-catenin mutant (β-cat/EnR) cannot undergo myogenic differentiation in the presence or absence of muscle-inducing levels of retinoic acid (RA). While RA could upregulate premyogenic mesoderm expression, including Pax3/7 and Meox1, only Pax3/7 and Gli2 could be upregulated by RA in the presence of β-cat/EnR. However, the use of a dominant negative construct that cannot be compensated by other factors is limiting due to the possibility of negative chromatin remodelling overriding compensatory mechanisms. In this study, we set out to determine if β-catenin function is essential for myogenesis with and without RA, by creating P19 cells with reduced β-catenin transcriptional activity using an shRNA approach, termed P19[shβ-cat] cells. The loss of β-catenin resulted in a reduction of skeletal myogenesis in the absence of RA as early as premyogenic mesoderm, with the loss of Pax3/7, Eya2, Six1, Meox1, Gli2, Foxc1/2, and Sox7 transcript levels. Chromatin immunoprecipitation identified an association of β-catenin with the promoter region of the Sox7 gene. Differentiation of P19[shβ-cat] cells in the presence of RA resulted in the upregulation or lack of repression of all of the precursor genes, on day 5 and/or 9, with the exception of Foxc2. However, expression of Sox7, Gli2, the myogenic regulatory factors and terminal differentiation markers remained inhibited on day 9 and overall skeletal myogenesis was reduced. Thus, β-catenin is essential for in vitro formation of premyogenic mesoderm, leading to skeletal myogenesis. RA can at least partially compensate for the loss of β-catenin in the expression of many myogenic precursor genes, but not for myoblast gene expression or overall myogenesis

Sox7 expression is directly modulated by Canonical Wnt signalling.

<p><i>Panel I:</i> P19 cells were differentiated in the presence or absence of 20 mM LiCl for 5 days. Changes in gene expression were analyzed by QPCR, normalized against GAPDH and represented as the average fold change over day 0 expression levels. <i>Panel II:</i> Conserved LEF/TCF binding elements (LBE) were identified by aligning DNA sequences from the mouse and human genomes using MULAN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057501#pone.0057501-Ovcharenko1" target="_blank">[44]</a>. <i>Panel III:</i> Crossed-linked chromatin was isolated from P19 cells on days 0 and 2 of DMSO-induced differentiation. ChIP was performed using an anti-β-catenin antibody, analyzed by QPCR with the indicated primers, and represented as the average percent of input sites that were immunoprecipitated. Error bars correspond to the ± SEM (n = 4). A one-way ANOVA was used to assess the statistical significance, where *p-value ≤0.05.</p

Skeletal myogenesis was reduced in P19[Shβ-cat] cells.

<p>Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO. Panel I: On day 9 of the differentiation, cells were fixed for immunofluorescence analysis using anti-Myosin heavy chain monoclonal antibodies (MHC; red) or anti-desmin antibodies (green), to visualize muscle cells and Hoechst dye to visualize cell nuclei (bar = 20 µm). Panel II: The degree of skeletal myogenesis was quantified by counting the number of MHC^+ve cells and expressed as the percentage of MHC^+ve cells in P19[shControl] cells. Error bars represent ± SEM (n = 6; 9000–11000 cells counted/condition). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.</p

Skeletal muscle, myoblast, and muscle precursor gene expression was reduced in P19[Shβ-cat] cells.

<p>Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO. RNA was harvested on days 0, 5 and 9 of the differentiation. QPCR was performed to quantify the transcript levels of the indicated genes. Changes in gene expression were normalized against β-actin and represented as fold change over day 0 transcript levels in P19[shControl] cells. Error bars correspond to the average ± SEM. The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.</p

Summary of changes in gene expression in P19[shβ-cat] cultures compared to same-day P19[shControl] cultures after differentiation in DMSO, with and without RA (− = decrease;+ = increase; NC = No change; NE = not expressed; ND = not determined; * = Enhanced or recovered gene expression due to RA treatment)(Data from Figs. 3 and 5).

<p>Summary of changes in gene expression in P19[shβ-cat] cultures compared to same-day P19[shControl] cultures after differentiation in DMSO, with and without RA (− = decrease;+ = increase; NC = No change; NE = not expressed; ND = not determined; * = Enhanced or recovered gene expression due to RA treatment)(Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057501#pone-0057501-g003" target="_blank">Figs. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057501#pone-0057501-g005" target="_blank">5</a>).</p

β-catenin transcriptional activity and protein levels were reduced in P19[shβ-cat] cells.

<p><i>Panel I:</i> P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO with or without 3 nM RA. β-catenin transcript levels were determined by QPCR, which was performed from RNA harvested on the days indicated (n = 12 for day 0, n = 5 for day 5, n = 4 for day 9). Changes in gene expression were normalized to β-actin and expressed as a percentage of P19[shControl] cells under each condition. Error bars represent ± SEM. Student’s t-test was used to assess statistical significance, with *p≤0.05. <i>Panel II&III</i>: β-catenin protein was detected by western blot analysis using protein isolated from the nuclei of P19[shControl] and P19[shβ-cat] cells differentiated for five days with DMSO. Relative β-catenin protein levels were quantified by densitometry and normalized to RNA polymerase II protein levels (loading control). Error bars represent ± SEM (n = 4). Panel IV: P19[shβ-cat] and P19[shControl] cells were aggregated for 1 day in the presence or absence of 20 mM LiCl. Firefly luciferase activity was measured from cells co-transfected with either Super8XTOPFLASH (TOP) or Super8XFOPFLASH (FOP) and the Renilla control construct. All firefly luciferase activity was normalized to Renilla and represented as the fold change over P19[shControl] FOP luciferase activity. Error bars correspond to the average ± SEM (n = 4). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05 was considered significant. Panel V: β-catenin protein was detected by western blot analysis using protein isolated from the nuclei of P19[shControl] and P19[shβ-cat] cells differentiated for one day with LiCl, as described in Panel II. Numbers represent quantification by densitometry of β-catenin protein levels, normalized to RNA Pol II.</p

RA can recover the expression of most skeletal muscle precursor genes in P19[shβ-cat] cells.

<p>Aggregated P19[shControl] and P19[shβ-cat] cells were differentiated in the presence of 1% DMSO with 3 nM RA. <i>Panels I & II:</i> RNA was harvested on days 0, 5 and 9 of the differentiation and analyzed by QPCR for all days (Panel I) or days 0 & 9 (Panel II). Changes in gene expression were normalized against β-actin represent fold change over day 0 levels in P19[shControl] cells. Error bars correspond to the ± SEM. Panel III: Cultures were fixed on day 9 for immunofluorescence with an anti-MHC antibody. The number of MHC^+ve cells were counted and expressed as a percentage of the control cells. Error bars represent ± SEM (n = 6). The Student’s t-test was used to assess statistical significance, where *p-value ≤0.05.</p

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C

Phosphorylation of the critical myogenic transcription factor MEF2C by skeletal MLCK promotes the recruitment of histone acetyltransferases to MEF2C-responsive promoters, thus regulating skeletal muscle gene expression and myoblast differentiation