title: Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.
abstract: Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.
authors.affiliation: Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.|New York State Center of Excellence in Bioinformatics and Life Sciences , 701 Ellicott Street, Buffalo, New York 14203, United States.
authors.forename: Chengjian
authors.initials: C
authors.lastname: Tu
authors.affiliation: Athenex Pharmaceuticals, 1001 Main Street, Suite 600, Buffalo, New York 14203, United States.
authors.forename: Yahao
authors.initials: Y
authors.lastname: Bu
authors.affiliation: Athenex Pharmaceuticals, 1001 Main Street, Suite 600, Buffalo, New York 14203, United States.
authors.forename: Marija
authors.initials: M
authors.lastname: Vujcic
authors.affiliation: Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.|New York State Center of Excellence in Bioinformatics and Life Sciences , 701 Ellicott Street, Buffalo, New York 14203, United States.
authors.forename: Shichen
authors.initials: S
authors.lastname: Shen
authors.affiliation: Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.|New York State Center of Excellence in Bioinformatics and Life Sciences , 701 Ellicott Street, Buffalo, New York 14203, United States.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.|Beijing University of Chinese Medicine , Beijing, 100029, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Qu
authors.affiliation: Athenex Pharmaceuticals, 1001 Main Street, Suite 600, Buffalo, New York 14203, United States.
authors.forename: David
authors.initials: D
authors.lastname: Hangauer
authors.affiliation: Athenex Pharmaceuticals, 1001 Main Street, Suite 600, Buffalo, New York 14203, United States.
authors.forename: James L
authors.initials: JL
authors.lastname: Clements
authors.affiliation: Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.|New York State Center of Excellence in Bioinformatics and Life Sciences , 701 Ellicott Street, Buffalo, New York 14203, United States.
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D006801:Humans; D008660:Metabolism / Q000187:drug effects; D024510:Muscle Development / Q000187:drug effects*; D032446:Myoblasts; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D011506:Proteins; D014409:Tumor Necrosis Factor-alpha
keywords: human muscle precursor cells; ion current-based quantification; myogenic differentiation; tumor necrosis factor