SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis

Du, Xiaohong; Yang, Liu; Liu, Lu; Cao, Qiuyu

doi:10.1038/s41419-020-2578-x

Cited by 56 publications

(38 citation statements)

References 69 publications

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“…In this study, we attempted to investigate the biological functions of SMAD4 in mammalian ovarian GCs by screening for its target genes and interacting coregulators. Bioinformatics analyses indicate that SMAD4 is crucial for the normal states and functions of GCs, as well as oocyte development and maturation, which is consistent with previous studies [ 23 , 24 ]. Additionally, the differentially expressed genes were identified, and we noticed that the expressions of multiple members in the TGF-β family signaling pathways were influenced in GCs after knockdown of SMAD4, especially the three important receptors (ACVR1B, BMPR2, and TGFBR2) described in this study, which highlighted the feedback regulation activities of SMAD4 in mammal GCs.…”

Section: Discussionsupporting

confidence: 91%

SMAD4 Feedback Activates the Canonical TGF-β Family Signaling Pathways

Liu

Yang

2021

IJMS

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TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs.

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Section: Discussionsupporting

confidence: 91%

SMAD4 Feedback Activates the Canonical TGF-β Family Signaling Pathways

Liu

Yang

2021

IJMS

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“…In this study, we have attempted to investigate the biological functions of SMAD4 in mammalian ovarian GCs by screening for its target genes and interacting proteins. Bioinformatics analyses indicate that SMAD4 is crucial for the normal states and functions of GCs, as well as oocyte development and maturation, which is consistent with the previous researches [22,28]. Besides, the differentially expressed genes were identi ed and interestingly found multiple members of TGF-β family signaling pathways including three important receptors (ACVR1B, BMPR2, and TGFBR2), which highlights the feedback regulation functions of SMAD4.…”

Section: Discussionsupporting

confidence: 87%

“…Fresh porcine bilateral ovaries were collected and placed in a thermos ask with 37°C PBS and transported back to the laboratory within 1 h. The collected ovaries were washed with PBS 5 times and porcine GCs were harvested from 2-5 mm non-atretic ovarian follicles by using syringe with 22-gauge needle and cultured as previously described [22]. Brie y, the isolated porcine GCs were washed with 37°C PBS containing 1% penicillin and streptomycin three times, and then seeded in cell culture plates with DEME/F12 containing 10% fetal bovine serum (FBS) in a 37°C humid atmosphere with 5% CO 2 .…”

Section: Cell Culture and Treatmentmentioning

confidence: 99%

SMAD4 Feedback Activates The Canonical TGF-β Family Signaling Pathways

Liu

2021

Preprint

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Background: TGF-β family signaling pathways, including TGF-β and BMP signaling pathways, are widely involved in the regulation of health and disease, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. The objective of this research is to investigate the molecular mechanism and function mode of SMAD4 directly feedback regulation of TGF-β family signaling pathways in porcine granulosa cells (GC).Results: The transcriptomic alteration of porcine GCs induced by SMAD4 silencing was re-analyzed with the background of Sus scrofa RefSeq 11.1 (Sscrofa 11.1). A total of 986 differentially expressed mRNAs (DEmRNAs) were identified, including 467 down-regulated and 519 up-regulated genes. Functional assessment showed the impacts of DEmRNAs on the regulation of states and function of GCs, and the oocyte development. As the upstream receptors of SMAD4, ACVR1B, BMPR2, and TGFBR2 were selected from down-regulated DEmRNAs for further research. In vitro, qRT-PCR and western blotting were performed and confirmed that SMAD4 significantly induced the expression of ACVR1B, BMPR2, and TGFBR2 in porcine GCs. Besides, RACE and luciferase activity assays were carried out to identified the core promoter of porcine ACVR1B, BMPR2, and TGFBR2. Results from ChIP assays showed that SMAD4 directly binds to the SMAD4 binding elements (SBEs) within the core promoter of its upstream receptors by acting as a transcription factor. Furthermore, c-JUN, CREB1, and SP1 were identified as SMAD4-interacted co-activators by IP assays and inhibition of which could dramatically suppress the expression of porcine ACVR1B, BMPR2, and TGFBR2 that induced by SMAD4 over-expression. Furthermore, three different interaction modes between SMAD4 and co-activators were identified by reciprocal ChIP assays.Conclusions: Take together, our findings revealed a novel feedback regulatory mechanism of TGF-β family signaling pathways in porcine GCs, and demonstrated for the first time that SMAD4, the only Co-SMAD, directly feedback activates the transcription of canonical TGF-β family signaling pathway receptors by interacting with three co-activators in different modes, which improves and expands the regulatory network, especially the feedback regulation modes of TGF-β family signaling pathways in the ovary.

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“…We identified several miRNAs differentially expressed between the patient groups which have not been previously associated with PCOS but are involved in the regulation of gene expression in follicles or in other ovary-related disorders. For example, hsa-miR-224-5p [60], which was up-regulated in the MGC of PCOS women in our study, downregulates SMAD4, which is involved in the regulation of apoptosis of granulosa cells [61]. It has been shown that hsa-miR-203a-3p, hsa-miR-195-5p, hsa-miR-486-3p, and hsa-miR-484 levels are altered in the granulosa cells of women with diminished ovarian reserve [24].…”

Section: Discussionsupporting

confidence: 45%

Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries

Rooda

Hasan

Roos

et al. 2020

IJMS

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Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

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SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis

Cited by 56 publications

References 69 publications

SMAD4 Feedback Activates the Canonical TGF-β Family Signaling Pathways

SMAD4 Feedback Activates the Canonical TGF-β Family Signaling Pathways

SMAD4 Feedback Activates The Canonical TGF-β Family Signaling Pathways

Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries

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