Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary
Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary
复制标题
DOI：
10.1210/en.138.12.5497
复制
发表时间：
1997-12-01
期刊：
影响因子：
4.8
通讯作者：
Hsueh, AJW
中科院分区：
文献类型：
--
作者：
Kaipia, A;Hsu, SY;Hsueh, AJW
bcl -2相关的抗和促凋亡蛋白在caspase蛋白酶上游的细胞内死亡程序的决定步骤中起重要作用。Bcl-2在转基因小鼠卵巢体细胞中靶向过表达，导致颗粒细胞凋亡减少，与排卵率升高、产仔数增加和卵巢畸胎瘤形成有关。外源性Bcl-2蛋白促进卵泡细胞存活的能力表明，该基因可以结合内源性卵巢Bcl-2家族成员，调节细胞内凋亡过程，有利于细胞存活。我们利用酵母双杂交系统寻找卵巢Bcl-2相互作用蛋白。筛选卵巢融合互补DNA文库获得了几个编码死亡激动剂Bcl-XL/ bcl -2相关死亡启动子（BAD）的克隆。Bcl-2同源结构域介导的Bcl-2相关蛋白的二聚化对其凋亡调节功能至关重要。与这些观察结果一致的是，酵母双杂交实验表明Bcl-2与BAD的相互作用依赖于Bcl-2的BH4和BH2结构域。Northern blot分析显示BAD信使RNA （mRNA）在不同组织中分布广泛，在肺、卵巢、子宫和大脑中含量最高。原位杂交分析显示，BAD mRNA在不同大小的卵泡颗粒细胞以及卵泡和间质细胞中均有表达。在出生后15-27天，BAD mRNA在卵巢中表达，在出生后第18天，即第一组早期窦卵泡形成时，BAD mRNA在发育发生的细胞凋亡中没有改变。同样，在未成熟大鼠雌激素停药引起的卵泡闭锁期间，BAD mRNA水平没有变化。为了研究BAD在卵巢中的作用，我们将BAD的互补DNA转染到颗粒细胞的原代培养物和性腺肿瘤细胞系中。BAD在两种细胞类型中过表达诱导凋亡，并且BAD的作用被一种膜渗透性caspase抑制剂逆转，这表明BAD通过激活caspase半胱氨酸蛋白酶诱导细胞凋亡。总之，死亡激动剂BAD被鉴定为卵巢中eel -a相互作用蛋白，BAD mRNA在颗粒细胞中组成性表达，表明BAD是卵巢细胞死亡过程的重要组成部分。由于BAD在颗粒细胞中过表达会导致细胞凋亡，未来对卵巢BAD结合蛋白及激素调控不同Bcl-2家族成员间相互作用的研究，将有助于更好地了解卵巢卵泡闭锁的细胞机制。
Bcl-2-related anti-and proapoptotic proteins are important in the decision step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the trans gene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Eel-a-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.