Makrigiannakis A, Coukos G, Christofidou-Solomidou M, Gour B J, Radice G L, Blaschuk O, Coutifaris C
Division of Human Reproduction, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelpha, PA, USA.
Am J Pathol. 1999 May;154(5):1391-406. doi: 10.1016/S0002-9440(10)65393-X.
Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-cadherin regulates human GC apoptosis via a cAMP-dependent mechanism. N-cadherin expression was evaluated in ovarian follicles and corpora lutea utilizing immunohistochemical techniques and in luteinized GCs in culture using immunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directed against both the extracellular and cytoplasmic domains of the molecule. Apoptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate of GC apoptosis was found to be two- to three-fold lower among aggregated cells, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadherin function disrupting antibodies or peptides exhibiting enhanced rates of apoptosis. GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea. N-cadherin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expression in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through cleavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular domain of N-cadherin and concomitantly inhibited the serum-deprivation-induced apoptosis of aggregated GCs. Collectively, these observations suggest that down-regulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevents cell aggregation and is associated with GC apoptosis. In addition, cAMP induces apoptosis in a dose-dependent manner, and this process is dependent, at least in part, on regulation of the N-cadherin molecule at the surface of the cells. We conclude that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.
研究表明，细胞间相互作用可能调节细胞凋亡，特别是钙依赖性细胞黏附分子N-钙黏蛋白已被证明能够调节这一过程。已知大鼠颗粒细胞（GCs）表达N-钙黏蛋白，而cAMP已知可诱导人和大鼠GCs凋亡。基于这些观察结果，我们推测N-钙黏蛋白通过cAMP依赖性机制调节人GCs凋亡。利用免疫组织化学技术在卵巢卵泡和黄体中评估N-钙黏蛋白表达，并在培养的黄体化GCs中使用免疫印迹、流式细胞术分析、免疫组织化学以及利用针对该分子细胞外和细胞质结构域的抗N-钙黏蛋白抗体的间接免疫荧光技术进行评估。通过TUNEL和DNA片段分析评估细胞凋亡，并通过流式细胞术细胞周期分析和电子显微镜进行确认。与单细胞相比，聚集细胞中的GCs凋亡率低两到三倍。发现聚集的体外培养GCs以及在存在破坏N-钙黏蛋白功能的抗体或肽的情况下培养的GCs中表达N-钙黏蛋白，而这些情况下GCs凋亡率增加。原位GCs在前 antral 和正常生长的排卵前卵泡以及早期黄体中对N-钙黏蛋白染色强烈。N-钙黏蛋白在闭锁卵泡和退化黄体中较弱。将GCs暴露于cAMP会增加细胞凋亡，同时以剂量依赖性方式降低N-钙黏蛋白蛋白表达。无血清条件下的细胞培养会增加细胞凋亡并降低N-钙黏蛋白表达，部分是通过切割该分子的细胞外结构域。金属蛋白酶抑制剂1,10-菲咯啉抑制N-钙黏蛋白细胞外结构域的切割，并同时抑制血清剥夺诱导的聚集GCs凋亡。总体而言，这些观察结果表明，N-钙黏蛋白的下调或该分子功能性细胞外结构域的缺失会阻止细胞聚集，并与GCs凋亡相关。此外，cAMP以剂量依赖性方式诱导细胞凋亡，并且这一过程至少部分取决于细胞表面N-钙黏蛋白分子的调节。我们得出结论，N-钙黏蛋白介导的GCs信号传导在卵泡和黄体细胞存活中起核心作用。
