Sun Yuhan, Liswaniso Simushi, Wu Hengsong, Sun Xue, Yan Chunchi, Qin Ning, Xu Rifu
Joint International Research Laboratory of Modern Agricultural Technology, Ministry of Education, Jilin Agricultural University, Changchun 130118, China.
Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China.
Genes (Basel). 2025 Feb 26;16(3):283. doi: 10.3390/genes16030283.
This study aims to investigate the regulation of small mothers against decapentaplegic 2 and 3 (Smad2/3) protein phosphorylation and nuclear exclusion in follicular granulosa cells (GCs) by chicken follicle-stimulating hormone (FSH) through the phosphatidylinositol 3-kinase (PI3K) signaling pathway, as well as the effect of Smad2/3 proteins on forkhead box O 3 and 4 (FoxO3/4). This lays the foundation for exploring the regulatory functions of signaling pathways closely related to follicular growth and development, as well as the molecular mechanisms of subcellular localization and nuclear exclusion of various effector factors (including transcription factors).
In this study, we used granulosa cells from 6-8 mm prehierachical follicles of chickens and performed immunofluorescence, quantitative real-time PCR (RT-qPCR), and Western blotting analysis to detect the phosphorylation and nuclear exclusion of Smad2/3 induced by FSH, as well as the regulatory effect of Smad2/3 on FOXO3/4 proteins.
The results showed that 10 ng/mL FSH and 50 μg/mL PI3K activator significantly reduced the phosphorylation level of Smad2/3 ( < 0.05), while no nuclear exclusion was observed. On the other hand, 16 nM/mL PI3K inhibitor and 50 μg/mL alkaline phosphatase significantly increased the phosphorylation level of Smad2/3 ( < 0.05). Overexpression of Smad2/3 increased the phosphorylation level of FOXO3/4 ( < 0.05); Smad2/3 interference resulted in a decrease in FOXO3/4 phosphorylation levels ( < 0.05).
FSH can inhibit Smad2/3 phosphorylation and retain it in the nucleus through the PI3K signaling pathway. Smad2/3 and FOXO3/4 act as downstream effectors of the PI3K signaling pathway, and Smad2/3 can promote the phosphorylation of FOXO3/4.
本研究旨在探讨鸡促卵泡激素（FSH）通过磷脂酰肌醇3-激酶（PI3K）信号通路对卵泡颗粒细胞（GCs）中抗五聚体蛋白2和3（Smad2/3）蛋白磷酸化及核排除的调控作用，以及Smad2/3蛋白对叉头框O 3和4（FoxO3/4）的影响。这为探索与卵泡生长发育密切相关的信号通路的调控功能，以及各种效应因子（包括转录因子）亚细胞定位和核排除的分子机制奠定了基础。
在本研究中，我们使用来自鸡6 - 8毫米等级前卵泡的颗粒细胞，进行免疫荧光、定量实时PCR（RT-qPCR）和蛋白质免疫印迹分析，以检测FSH诱导的Smad2/3磷酸化和核排除，以及Smad2/3对FOXO3/4蛋白的调控作用。
结果显示，10 ng/mL FSH和50 μg/mL PI3K激活剂显著降低了Smad2/3的磷酸化水平（<0.05），但未观察到核排除现象。另一方面，16 nM/mL PI3K抑制剂和50 μg/mL碱性磷酸酶显著增加了Smad2/3的磷酸化水平（<0.05）。Smad2/3的过表达增加了FOXO3/4的磷酸化水平（<0.05）；Smad2/3干扰导致FOXO3/4磷酸化水平降低（<0.05）。
FSH可通过PI3K信号通路抑制Smad2/3磷酸化并使其保留在细胞核内。Smad2/3和FOXO3/4作为PI3K信号通路的下游效应因子，且Smad2/3可促进FOXO3/4的磷酸化。
