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Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

ABSTRACT: The balance of myogenic and adipogenic differentiation is crucial for skeletal muscle homeostasis. Given the vital role of membrane proteins (MBPs) in cell signal perception, membrane proteomics was conducted to delineate mechanisms regulating differentiation of adipogenic and myogenic precursors in skeletal muscle. Adipogenic and myogenic precursors with divergent differentiation potential were isolated from the longissimus dorsi muscle of neonatal pigs by the preplate method. A total of 85 differentially expressed MBPs ( P < 0.05 and fold change ≥1.2 or ≤0.83) between 2 precursors were detected via isobaric tags for relative and absolute quantitation (iTRAQ) assay, including 67 up-regulated and 18 down-regulated in myogenic precursors. Functional enrichment analysis uncovered that myogenic and adipogenic precursors showed significant differences in cytoskeleton organization, syncytium formation, environmental information processing, and organismal systems. Furthermore, key MBPs in regulating cell differentiation were also characterized, including ITGB3, ITGAV, ITPR3, and EPHA2. Noteworthily, EPHA2 was required for myogenic differentiation, and it may promote myogenic differentiation through ERK signaling. Collectively, our study provided an insight into the distinct MBP profile between myogenic and adipogenic precursors in skeletal muscle and served as a solid basis for supporting the role of MBPs in regulating differentiation.-Zhang, X., Wang, L., Qiu, K., Xu, D., Yin, J. Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

SUBMITTER: Zhang X

PROVIDER: S-EPMC6436648 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSkeletal muscle is a complex and heterogeneous tissue accounting for approximately 40% of body weight. Excessive ectopic lipid accumulation in the muscle fascicle would undermine the integrity of skeletal muscle in humans but endow muscle with marbling-related characteristics in farm animals. Therefore, the balance of myogenesis and adipogenesis is of great significance for skeletal muscle homeostasis. Significant DNA methylation occurs during myogenesis and adipogenesis; however, DNA methylation pattern of myogenic and adipogenic precursors derived from skeletal muscle remains unknown yet.MethodsIn this study, reduced representation bisulfite sequencing was performed to analyze genome-wide DNA methylation of adipogenic and myogenic precursors derived from the skeletal muscle of neonatal pigs. Integrated analysis of DNA methylation and transcription profiles was further conducted. Based on the results of pathway enrichment analysis, myogenic precursors were transfected with CACNA2D2-overexpression plasmids to explore the function of CACNA2D2 in myogenic differentiation.ResultsAs a result, 11,361 differentially methylated regions mainly located in intergenic region and introns were identified. Furthermore, 153 genes with different DNA methylation and gene expression level between adipogenic and myogenic precursors were characterized. Subsequently, pathway enrichment analysis revealed that DNA methylation programing was involved in the regulation of adipogenic and myogenic differentiation potential through mediating the crosstalk among pathways including focal adhesion, regulation of actin cytoskeleton, MAPK signaling pathway, and calcium signaling pathway. In particular, we characterized a new role of CACNA2D2 in inhibiting myogenic differentiation by suppressing JNK/MAPK signaling pathway.ConclusionsThis study depicted a comprehensive landmark of DNA methylome of skeletal muscle-derived myogenic and adipogenic precursors, highlighted the critical role of CACNA2D2 in regulating myogenic differentiation, and illustrated the possible regulatory ways of DNA methylation on cell fate commitment and skeletal muscle homeostasis.

Project description:All-trans-retinoic acid (atRA) is an essential signaling molecule in embryonic development. It regulates cell differentiation by activating nuclear retinoic acid receptors (RAR) and retinoid-X receptors (RXR), which both control gene expression. In addition, atRA could act in the cytoplasm by modulating the activity of mitogen-activated protein kinases (MAPK) ERK and p38, which also have a role in cell differentiation. AtRA can induce the differentiation of P19 embryonic carcinoma stem cells into adipocytes, cardiomyocytes, and skeletal muscle cells, concurrently, in the same culture. We postulated that combinations of atRA, atRA analogs exhibiting selectivity for RAR or RXR, and inhibitors of ERK and p38 signaling (ERKi and p38i) could be used to favor one mesodermal fate over the others in the P19 model. In a first series of experiments, we replaced atRA by an agonist of RXR (LG100268) or RAR (TTNPB) to preferentially stimulate one group of receptors over the other. LG100268 was as adipogenic and myogenic as atRA, whereas TTNPB strongly induced adipogenesis, but not myogenesis. ERKi enhanced the myogenic action of atRA, and p38i increased both adipogenesis and myogenesis. In a second series of experiments, we combined atRA with an RAR or RXR antagonist (RARatg or RXRatg) to preferentially deactivate each receptor group in turn. The combinations atRA+RXRatg and atRA+RARatg, including or not ERKi, had similar mesodermal actions as atRA. In contrast, there was no myogenesis with atRA+RXRatg+p38i treatment, and there were no myogenesis and no adipogenesis with the atRA+RARatg+p38i combination. Overall, the results indicate that p38 has a role in mesodermal differentiation that depends on the retinoid context. Indeed, p38 in conjunction with RXR is important in myogenesis, and p38 and RAR in adipogenesis. Under the conditions tested, it was possible to stimulate adipogenesis with a block on myogenesis, whereas increased myogenesis was accompanied by adipogenesis.

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Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling.

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