Caron Pierre-Luc, Fréchette-Frigon Guylaine, Shooner Carl, Leblanc Valérie, Asselin Eric
Départment de Chimie-Biologie, Groupe de Recherche en Biopathologies Cellulaires et Moléculaires, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, G9A 5H7, Canada.
Reprod Biol Endocrinol. 2009 Aug 5;7:80. doi: 10.1186/1477-7827-7-80.
During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-beta isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.
Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1-9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms.
Our results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner.
Those results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.
在发情周期中，大鼠子宫内膜会经历许多变化，如细胞增殖和凋亡。如果发生着床，基质细胞会分化为蜕膜细胞，在妊娠末期会发生第二轮凋亡。这个过程称为蜕膜退化，受到严格调控，因为它对成功妊娠至关重要。我们之前已经表明，转化生长因子β1（TGF-β1）、转化生长因子β2（TGF-β2）和转化生长因子β3（TGF-β3）在基底蜕膜退化期间的子宫内膜中表达，但尽管我们已经证明TGF-β1参与了蜕膜细胞凋亡的调控，但TGF-β2和TGF-β3亚型在这些细胞中触发凋亡机制的能力仍然未知。此外，我们推测TGF-βs在发情周期的非妊娠子宫内膜中也存在并受到调控。本研究的目的是确定并比较每种TGF-β亚型在体外对致敏子宫内膜基质细胞凋亡调控中的具体作用，并研究体内发情周期中子宫内膜中TGF-β亚型的调控情况。
将发情周期规律（4天）的大鼠在发情周期的不同天数（动情后期、动情前期、动情期和动情间期）处死。用性类固醇诱导去卵巢大鼠假孕，并在不同天数（第1 - 9天）处死大鼠。收集子宫，要么固定用于免疫组织化学染色（IHC），要么进行逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析。对于研究的体外部分，将大鼠去卵巢并用性类固醇诱导蜕膜化。纯化、培养子宫内膜基质蜕膜细胞，并用不同浓度的TGF-β亚型处理。
我们的结果表明，所有三种TGF-β亚型均存在，但在发情周期中在子宫内膜中的定位不同，并且在假孕期间它们的表达调控也不同。在培养的基质细胞中，我们发现TGF-β3亚型诱导Smad2磷酸化，表明Smad信号通路在这些细胞中被TGF-β3激活。此外，如Hoechst核染色所示，TGF-β2和TGF-β3在培养的基质细胞中诱导凋亡呈剂量依赖性增加。值得注意的是，TGF-β2和TGF-β3以剂量依赖的方式降低了抗凋亡蛋白XIAP的水平以及磷酸化/活性Akt（一种众所周知的说存活蛋白）的水平。
这些结果表明，TGF-β可能在发情周期中子宫内膜重塑以及大鼠蜕膜细胞凋亡调控中起重要作用，其中抑制Akt存活信号通路可能是参与凋亡调控的重要机制。
