Chang Hsun-Ming, Cheng Jung-Chien, Fang Lanlan, Qiu Xin, Klausen Christian, Taylor Elizabeth L, Leung Peter C K
Department of Obstetrics and Gynaecology, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
J Clin Endocrinol Metab. 2015 Mar;100(3):E365-74. doi: 10.1210/jc.2014-2496. Epub 2014 Dec 16.
Theca cell-derived bone morphogenetic protein 4 (BMP4) and BMP7 are important regulators of folliculogenesis and have been shown to inhibit luteinization. Pentraxin 3 (PTX3) plays a critical role in the assembly of the cumulus oophorus extracellular matrix, which is essential for cumulus expansion during ovulation and may be modulated by BMP4 and BMP7.
The aim of this study was to investigate the effects of BMP4 and BMP7 on the expression of PTX3 in human granulosa cells and to examine their underlying molecular determinants.
An established immortalized human granulosa cell line (SVOG), a granulosa cell tumor cell line (KGN), and primary granulosa-lutein cells were used as study models. PTX3 expression and accumulation as well as Smad1/5/8 phosphorylation were examined after exposure to recombinant human BMP4 and BMP7. BMP type I receptor involvement was investigated with inhibitors (dorsomorphin and DMH-1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline)) and small interfering RNAs targeting activin receptor-like kinase (ALK)2, ALK3, and/or ALK6. Small interfering RNAs targeting Smad4 were used to verify the involvement of Smad signaling.
The study was conducted at an academic research center.
Quantitative RT-PCR and Western blot were used to measure mRNA and protein levels, respectively. Levels of PTX3 and BMP4 were measured by ELISA.
Treatment with BMP4 and BMP7 significantly decreased PTX3 mRNA and protein production. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by cotreatment with 2 BMP type I receptor inhibitors (dorsomorphin and/or DMH-1). Combined knockdown (ALK3/ALK6 for BMP4 and ALK2/ALK3 for BMP7) reversed the effects of BMP4- and BMP7-induced Smad1/5/8 phosphorylation and PTX3 suppression. Furthermore, Smad4 knockdown reversed the suppressive effects of BMP4 and BMP7 on PTX3 expression. In follicular fluid, concentrations of PTX3 were negatively correlated with concentrations of BMP4.
BMP4 and BMP7 use differential subsets of BMP type I receptors to downregulate PTX3 expression via Smad-dependent signaling in human granulosa cells.
卵泡膜细胞衍生的骨形态发生蛋白4（BMP4）和BMP7是卵泡发生的重要调节因子，已被证明可抑制黄体化。五聚体3（PTX3）在卵丘细胞外基质的组装中起关键作用，这对于排卵期间的卵丘扩展至关重要，并且可能受BMP4和BMP7调节。
本研究旨在探讨BMP4和BMP7对人颗粒细胞中PTX3表达的影响，并研究其潜在的分子决定因素。
使用已建立的永生化人颗粒细胞系（SVOG）、颗粒细胞瘤细胞系（KGN）和原代颗粒黄体细胞作为研究模型。在暴露于重组人BMP4和BMP7后，检测PTX3的表达和积累以及Smad1/5/8磷酸化。使用抑制剂（多索茶碱和DMH-1（4-[6-[4-(1-甲基乙氧基)苯基]吡唑并[1,5-a]嘧啶-3-基]-喹啉））和靶向激活素受体样激酶（ALK）2、ALK3和/或ALK6的小干扰RNA研究BMP I型受体的参与情况。使用靶向Smad4的小干扰RNA来验证Smad信号通路的参与情况。
该研究在一个学术研究中心进行。
分别使用定量RT-PCR和蛋白质印迹法测量mRNA和蛋白质水平。通过ELISA测量PTX3和BMP4的水平。
用BMP4和BMP7处理显著降低了PTX3 mRNA和蛋白质的产生。这些抑制作用以及Smad1/5/8磷酸化的诱导，通过与两种BMP I型受体抑制剂（多索茶碱和/或DMH-1）共同处理而减弱。联合敲低（BMP4的ALK3/ALK6和BMP7的ALK2/ALK3）逆转了BMP4和BMP7诱导的Smad1/5/8磷酸化和PTX3抑制的作用。此外，Smad4敲低逆转了BMP4和BMP7对PTX3表达的抑制作用。在卵泡液中，PTX3的浓度与BMP4的浓度呈负相关。
BMP4和BMP7在人颗粒细胞中通过Smad依赖的信号通路，利用不同的BMP I型受体亚群下调PTX3的表达。
