TRAF4 acts as a fate checkpoint to regulate the adipogenic differentiation of MSCs by activating PKM2

Abstract

Background: Mesenchymal stem cells (MSCs) selectively differentiate into adipocytes or osteoblasts, and several molecules control the fate determination of MSCs. Understanding these key checkpoints greatly contributes to the ability to induce specific MSC differentiation for clinical applications. In this study, we aimed to explore whether TNF receptor-associated factor 4 (TRAF4) affects MSC adipogenic differentiation, which we previously reported that could positively regulated the osteogenic differentiation. Methods: Western blotting and Real-time Polymerase Chain Reaction were used to detected the expression pattern of TRAF4 during adipogenic differentiation. Lentivirus was constructed to regulate TRAF4 expression, and oil red O staining and Western blotting were used to assess its role in adipogenesis, which was confirmed in vivo by implanting an MSC-matrigel mixture into nude mice. Western blotting was used to detect the activated signaling pathways, and a specific inhibitor and agonist were used to clear the roles of the key signaling pathways. Additionaly, Co-Immunoprecipitation was conducted to find that Pyruvate kinase isozyme type M2 (PKM2) interacts with TRAF4, and to further explore their binding and functional domains. Finally, an RNA-binding protein immunoprecipitation assay and Western blotting were used to detect whether N6methyladenosine mediates the decreased TRAF4 expression during adipogenic differentiation. Findings: The results demonstrated that TRAF4 negatively regulates MSC adipogenesis in vitro and in vivo. Mechanistically, we revealed that TRAF4 binds to PKM2 to activate the kinase activity of PKM2, which subsequently activates b-catenin signaling and then inhibits adipogenesis. Furthermore, TRAF4 downregulation during adipogenesis is regulated by ALKBH5-mediated N6-methyladenosine RNA demethylation. Interpretation: TRAF4 negatively regulates the adipogenesis of MSCs by activating PKM2 kinase activity, which may act as a checkpoint to fine-tune the balance of adipo-osteogenic differentiation, and suggests that TRAF4 may be a novel target of MSCs in clinical use and may also illuminate the underlying mechanisms of bone metabolic diseases.

Curcumin prevents obesity by targeting TRAF4‐induced ubiquitylation in m 6 A‐dependent manner

“…Since ubiquitination plays crucial roles in the onset and progression of cancer and metabolic syndromes (Popovic et al, 2014), we speculate that there might exist a correlation between ALKBH5 and ubiquitination in the regulation of physiological processes. In this regard, our current work links with reminiscent of a recent study that showed the TRAF4 expression levels were positively correlated with ALKBH5 in MSC (Cen et al, 2020). On the contrary, our result showed that the TRAF4 negatively correlated with ALKBH5 expression in 3T3-L1 cells.…”

Long noncoding RNA LYPLAL1-AS1 regulates adipogenic differentiation of human mesenchymal stem cells by targeting desmoplakin and inhibiting the Wnt/β-catenin pathway

“…This makes it difficult for us to generate conditional knockout mice to study the effects of LYPLAL1-AS1 on adipogenesis in vivo. Based on the results from our study and other studies 52 , 53 , in vivo subcutaneous Matrigel plugs are a comparatively feasible method for testing the de novo adipogenesis ability of a lncRNA.…”

Caffeic and Chlorogenic Acids Synergistically Activate Browning Program in Human Adipocytes: Implications of AMPK- and PPAR-Mediated Pathways

“…Repressive PPARγ modulators acting as inverse agonists are suggested as promising therapeutic options in obesity management and are associated with enhancement in bone formation and improved WAT metabolic functions [ 9 , 48 , 59 , 60 ]. Furthermore, recent insights into the structural mechanisms of the PPARγ repressive function [ 48 ] permit better understanding of the interpretation of the interactions between chemical ligands and PPARγ protein.…”

“…Considering the limited number of clinically available anti-obesity drugs [ 2 ], finding promising candidates for novel drug leads is on urgent demand. Development of effective treatments counteracting obesity is directed to several, indeed, overlapping approaches: (i) obstruction of excessive lipid accumulation and adipocyte hypertrophy [ 3 , 4 , 5 , 6 ]; (ii) resolution of the chronic inflammation in white adipose tissue (WAT) of the obese [ 7 , 8 , 9 ]; (iii) induction of metabolically healthy adipocyte recruitment [ 10 , 11 , 12 ]; (iv) promotion of energy expenditure through enhanced lipolysis, thermogenesis and browning of the WAT [ 13 , 14 , 15 , 16 , 17 , 18 ].…”