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[Tribbles pseudokinase 3 inhibits the adipogenic differentiation of human adipose-derived mesenchymal stem cells]
1Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Objective:
To identify the role of Tribbles pseudokinase 3 (TRIB3) during the process of adipogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs), and to provide a new target and a novel idea for the application of hASCs in adipose tissue engineering and soft tissue regeneration.
Methods:
TRIB3-knockdown hASCs (shTRIB3) and TRIB3-overexpression hASCs (TRIB3-over) were established using lentivirus transfection technique. The transfection effect was estimated by the visible presence of green fluorescence as the expression of green fluorescent protein (GFP) in the transfected hASCs. The lentiviral transfection efficiency was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. After adipogenic induction, Oil Red staining and quantification, as well as qRT-PCR about several specific adipogenic markers were used to evaluate the adipogenic differentiation ability of hASCs.
Results:
In TRIB3-knockdown hASCs, the TRIB3 mRNA expression level decreased by about 84.3% compared with the control group (P<0.01), and the TRIB3 protein level also showed obvious reduction. Oppositely, in TRIB3-overexpression hASCs, the TRIB3 mRNA expression level increased by approximately 160% compared with the control group (P<0.01), and the TRIB3 protein level also showed a significant increase. These results indicated a successful construction of TRIB3-knockdown hASCs and TRIB3-overexpression hASCs. The Oil Red staining results showed that the down-regulation of TRIB3 significantly promoted lipid droplets formation in hASCs, consistent with Oil Red quantification. On the other hand, the up-regulation of TRIB3 suppressed lipid droplets formation in hASCs, consistent with Oil Red quantification. After adipogenic induction, adipogenesis-related genes, including peroxisome proliferator-activated receptor γ (PPARγ), cluster of differentiation 36 (CD36) and CCAAT/enhancer binding protein α (C/EBPα), were increased significantly in TRIB3-knockdown hASCs compared with the control group (P<0.01). Oppositely, PPARγ, CD36 and lipoprotein lipase (LPL) were significantly decreased in TRIB3-overexpression hASCs compared with the control group (P<0.01).
Conclusion:
TRIB3 inhibited the adipogenic differentiation of hASCs. Knockdown of TRIB3 promoted the ability of adipogenesis of hASCs, while overexpression of TRIB3 inhibited the adipogenic differentiation of hASCs. Considering the important role of PPARγ in the adipogenis process, the molecular mechanism of the regulatory function of TRIB3 may be related with PPARγ signal pathway.
Insights
Tribbles pseudokinase 3 (TRIB3) inhibits the differentiation of human adipose-derived mesenchymal stem cells (hASCs) into fat cells. Reducing TRIB3 enhances adipogenesis, offering potential for tissue engineering.
Area of Science:
- Stem Cell Biology
- Molecular Biology
- Tissue Engineering
Background:
- Human adipose-derived mesenchymal stem cells (hASCs) are crucial for adipose tissue engineering and soft tissue regeneration.
- Understanding the molecular regulation of hASC adipogenesis is vital for optimizing these applications.
Purpose of the Study:
- To elucidate the role of Tribbles pseudokinase 3 (TRIB3) in the adipogenic differentiation of hASCs.
- To identify TRIB3 as a potential therapeutic target for enhancing hASC-based regenerative strategies.
Main Methods:
- Lentivirus-mediated knockdown (shTRIB3) and overexpression (TRIB3-over) of TRIB3 in hASCs.
- Assessment of transfection efficiency using qRT-PCR and Western blot.
- Evaluation of adipogenic differentiation via Oil Red staining and quantification of key adipogenic marker genes (PPARγ, CD36, C/EBPα, LPL).
Main Results:
- Successful generation of TRIB3-knockdown and TRIB3-overexpressing hASCs confirmed by qRT-PCR and Western blot.
- TRIB3 knockdown significantly promoted lipid droplet formation and upregulated adipogenic genes (PPARγ, CD36, C/EBPα).
- TRIB3 overexpression suppressed lipid droplet formation and downregulated adipogenic genes (PPARγ, CD36, LPL).
Conclusions:
- TRIB3 acts as an inhibitor of hASC adipogenic differentiation.
- Modulating TRIB3 levels offers a novel strategy to control hASC differentiation for therapeutic applications.
- The inhibitory mechanism of TRIB3 likely involves the PPARγ signaling pathway.
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