Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells
Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells
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DOI：
10.1093/humrep/dev175
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发表时间：
2015-09-01
影响因子：
6.1
通讯作者：
Leung, Peter C. K.
中科院分区：
文献类型：
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作者：
Chen, Yu-Ching;Chang, Hsun-Ming;Leung, Peter C. K.
转化生长因子-β1是否上调人颗粒细胞连接蛋白43(Cx43)，促进细胞间通讯？转化生长因子-β1上调人颗粒细胞间隙连接通讯活性(GJIC)，这种作用是通过激活素受体样激酶(ALK)5介导的Sma和Mad相关蛋白(SMAD)2/3-Smad4依赖的途径实现的。在人类颗粒细胞中，Cx43缝隙连接在卵泡和卵母细胞的发育中起着重要的作用。本实验历时一年，以永生化的人颗粒细胞(SVOG细胞)和原代培养的人颗粒黄体细胞为研究模型。用激活素/转化生长因子-βI型受体抑制剂SB431542和针对ALK4、Alk5、Smad2、Smad3和Smad4的小干扰RNA来验证作用的特异性并探讨其分子机制。实时荧光定量聚合酶链式反应和蛋白质印迹分析分别检测特异性mRNA和蛋白水平。细胞间GJIC的检测采用刮擦负载和染料转移实验。结果用单因素方差分析，转化生长因子-β1处理增加Smad2/3的磷酸化(P<；0.0001)和上调Cx43mRNA和蛋白水平(P<；0.001)，这些刺激作用被转化生长因子-βI型受体抑制剂SB431542所阻断。此外，在原代培养的人颗粒黄体细胞中，证实了转化生长因子-β1对Cx43表达(mRNA和蛋白)的上调作用(P<；0.05)。小干扰RNA介导的Alk5的下调，而不是Alk4的下调，阻断了转化生长因子-β1诱导的Smad2/3的磷酸化和Cx43的上调。此外，Smad2/3或常见的Smad4基因的敲除可取消转化生长因子-β1对SVOG细胞Cx43表达的刺激作用。转化生长因子-β1诱导的Cx43表达上调导致卵巢颗粒细胞间GJIC的增加(P<；0.001)。本研究的结果来源于体外系统，可能不能反映卵巢内的微环境。我们的研究首次全面研究了转化生长因子-β1调控人颗粒细胞Cx43表达和GJIC的分子机制，证明转化生长因子-β1可能在细胞-细胞通讯的局部调控中起关键作用。加深对分子决定因素的了解将为卵巢生理学提供重要的见解，并有助于开发潜在的生育调节治疗方法。
Does transforming growth factor-beta 1 (TGF-beta 1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells?TGF-beta 1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway.TGF-beta 1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-beta 1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes.This is an experimental study which was performed over a 1-year period.Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-beta 1. An activin/TGF-beta type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.TGF-beta 1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-beta type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-beta 1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-beta 1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-beta 1 on Cx43 expression in SVOG cells. The TGF-beta 1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001).The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo.Our studies represent the first comprehensive research of molecular mechanisms of TGF-beta 1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-beta 1 may play a crucial role in the local modulation of cell-cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation.