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SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis
Xing Du1, Zengxiang Pan1, Qiqi Li1
1College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
Abstract:
Canonical TGF-β signals are transduced from the cell surface to the cytoplasm, and then translocated into the nucleus, a process that involves ligands (TGF-β1), receptors (TGFBR2/1), receptor-activated SMADs (SMAD2/3), and the common SMAD (SMAD4). Here we provide evidence that SMAD4, a core component of the canonical TGF-β signaling pathway, regulates the canonical TGF-β signaling pathway in porcine granulosa cells (GCs) through a feedback mechanism. Genome-wide analysis and qRT-PCR revealed that SMAD4 affected miRNA biogenesis in GCs. Interestingly, TGFBR2, the type II receptor of the canonical TGF-β signaling pathway, was downregulated in SMAD4-silenced GCs and found to be a common target of SMAD4-inhibited miRNAs. miR-425, the most significantly elevated miRNA in SMAD4-silenced GCs, mediated the SMAD4 feedback regulation of the TGF-β signaling pathway. This was accomplished through a direct interaction between the transcription factor SMAD4 and the miR-425 promoter, and a direct interaction between miR-425 and the TGFBR2 3'-UTR. Furthermore, miR-425 enhanced GC apoptosis by targeting TGFBR2 and the canonical TGF-β signaling pathway, which was rescued by SMAD4 and TGF-β1. Overall, our findings demonstrate that a positive feedback mechanism exists within the canonical TGF-β signaling pathway. This study also provides new insights into mechanism underlying the canonical TGF-β signaling pathway, which regulates GC function and follicular development.
Insights
SMAD4 regulates the TGF-β signaling pathway in porcine granulosa cells via a feedback loop involving miR-425 and TGFBR2. This mechanism impacts cell apoptosis and follicular development.
Area of Science:
- Cell Biology
- Molecular Biology
- Endocrinology
Background:
- Canonical transforming growth factor-beta (TGF-β) signaling is crucial for cellular processes.
- SMAD4 is a key transcription factor in the TGF-β pathway.
- Granulosa cells (GCs) play vital roles in ovarian function and follicular development.
Purpose of the Study:
- To investigate the role of SMAD4 in regulating the canonical TGF-β signaling pathway in porcine granulosa cells.
- To elucidate the feedback mechanism involving SMAD4, microRNAs (miRNAs), and TGF-β receptors.
- To understand the impact of this pathway on GC apoptosis and follicular development.
Main Methods:
- Genome-wide analysis and quantitative real-time PCR (qRT-PCR) to assess gene expression.
- SMAD4 silencing to observe downstream effects.
- miRNA profiling and analysis.
- Luciferase reporter assays to confirm interactions between SMAD4, miR-425, and TGFBR2.
Main Results:
- SMAD4 silencing affected miRNA biogenesis in GCs.
- Transforming growth factor-beta receptor 2 (TGFBR2) was downregulated in SMAD4-silenced GCs and targeted by SMAD4-inhibited miRNAs.
- miR-425, upregulated in SMAD4-silenced GCs, directly interacted with the miR-425 promoter and TGFBR2 3'-UTR, mediating feedback regulation.
- miR-425 promoted GC apoptosis by targeting TGFBR2 and the TGF-β pathway, effects rescued by SMAD4 and TGF-β1.
Conclusions:
- A positive feedback mechanism exists within the canonical TGF-β signaling pathway in porcine GCs, involving SMAD4, miR-425, and TGFBR2.
- This feedback loop regulates GC apoptosis and influences follicular development.
- The findings provide novel insights into the molecular mechanisms governing GC function and ovarian physiology.
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