SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis

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.

Reduced exosome miR‐425 and miR‐744 in the plasma represents the progression of fibrosis and heart failure

“…Xing Du et al reported that SMAD4, a downstream protein of TGF-b pathway, control the expression of miR-425 by binding to the promoter region. Meanwhile, miR-425 directly repressed TGFBR2 expression via targeting 3 0 UTR and functioned as a feedback [20]. Our research reported that TGF-b is a target of miR-425 for the first time, and we confirmed, in the human cardiac fibroblasts, that TGF-b protein level was repressed by miR-425 and miR-744.…”

NORFA, long intergenic noncoding RNA, maintains sow fertility by inhibiting granulosa cell death

“…6a). Of them, TGFBR2 is an important component of TGF-β signaling pathway that is involved in porcine granulosa cell apoptosis and follicular atresia 31 . MRE motif of miR-126 was located at the residues 484-506 nt in the porcine TGFBR2 3′-untranslated region (UTR).…”

“…Western blotting. Western blotting assays were performed as described previously 31 . The primary antibodies used here are anti-TGFBR2 (Sangon Biotech, #D155818, 1:1000), anti-SMAD3 (Sangon Biotech, #D155234, 1:1000), anti-p-SMAD3 (Sangon Biotech, #D155153, 1:1000), anti-NFIX (Affinity, #DF3250, 1:1000), and anti-GAPDH (ORIGENE, #TA802519, 1:3000).…”

“…Chromatin immunoprecipitation. ChIP assays were performed as previously described 31 . Briefly, Rabbit anti-NFIX (Affinity, #DF3250) antibody was used to pull down NFIX-DNA complex and a nonspecific antibody against IgG (Santa Cruz, #sc-2358) was used as an internal control, and the chromatin before immunoprecipitation was used as an input control.…”

MIR-361-5P Mediates TGF-β Signalling to Promote Granulosa Cell Apoptosis Through Vegfa During Porcine Follicle Atresia

“…The successful mutations were identi ed by sequencing technology. The overexpression plasmid pcDNA3.1-SMAD4 plasmid was generated previously by our group (Du et al, 2018). Primers used here are detailed in Supplementary Table S3.…”