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Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo
Published on: July 20, 2019
Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on
Chengjian Tu1,2, Yahao Bu3, Marija Vujcic3
1Department of Pharmaceutical Sciences, State University of New York at Buffalo , 285 Kapoor Hall, Buffalo, New York 14260, United States.
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.
Insights
Tumor necrosis factor-alpha (TNF-α) disrupts muscle cell differentiation by altering key proteins and signaling pathways. This proteomic study reveals mechanisms for potential therapeutic interventions in muscle wasting conditions.
Area of Science:
- Muscle biology
- Cellular signaling
- Proteomics
Background:
- Tumor necrosis factor-alpha (TNF-α) is implicated in muscle wasting and cachexia.
- The molecular mechanisms by which TNF-α disrupts myoblast differentiation are not fully understood.
Purpose of the Study:
- To investigate the molecular mechanisms of TNF-α-regulated myogenic differentiation using proteomic profiling.
- To identify key signaling pathways and molecular players involved in TNF-α-induced dysregulation of muscle precursor cell (MPC) differentiation.
Main Methods:
- Comparative proteomic analysis of primary human MPCs cultured in growth medium, differentiation medium, and differentiation medium with TNF-α.
- Utilized an ion current-based quantitative proteomic platform with nano-LC separation.
- Validated findings through immunoassay analysis and assessment of myogenic markers (MYOD, myogenin) and MHC class I components.
Main Results:
- TNF-α inhibited myogenic differentiation, evidenced by reduced myotube formation and altered expression of MYOD and myogenin.
- NF-κB and STAT protein cooperation was identified as a key mechanism for TNF-α-induced differentiation dysregulation.
- Increased expression of MHC class I components (HLA-A, B, C, beta-2-microglobulin) was observed.
- Cholesterol biosynthesis inhibition during differentiation was not rescued by TNF-α, suggesting it's an early myogenesis event.
Conclusions:
- Proteomic profiling reveals TNF-α alters transcription regulators, metabolic processes, and signaling pathways in MPCs during myogenic differentiation.
- Findings provide insights into the molecular basis of TNF-α-mediated muscle wasting.
- Identified potential therapeutic targets and strategies, including the role of cholesterol metabolism and statins, for myogenic cellular therapeutics.
