Peluso J J, Pappalardo A, Fernandez G
Departments of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Endocrinology. 2001 Oct;142(10):4203-11. doi: 10.1210/endo.142.10.8460.
Previous studies have demonstrated that basic fibroblast growth factor prevents granulosa cell apoptosis. The following six observations provide insight into the mechanism by which basic fibroblast growth factor mediates its antiapoptotic action. First, loading granulosa cells with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator, prevented apoptosis when granulosa cells were deprived of basic fibroblast growth factor. Second, treatment with thapsigargin, an agent known to increase intracellular free calcium, induced granulosa cell apoptosis even in the presence of basic fibroblast growth factor. Third, an activator of PKC mimicked, whereas PKC inhibitors blocked, basic fibroblast growth factor's antiapoptotic action. Fourth, continuous basic fibroblast growth factor exposure maintained relatively constant levels of intracellular free calcium, and a PKC inhibitor induced a sustained 2- to 3-fold increase in intracellular free calcium. Fifth, granulosa cells, as well as spontaneously immortalized granulosa cells, were shown to express PKC delta, -lambda, and -zeta. Finally, the PKC delta-specific inhibitor, rottlerin, blocked basic fibroblast growth factor's antiapoptotic action in granulosa cells and spontaneously immortalized granulosa cells. These studies suggest that basic fibroblast growth factor regulates intracellular free calcium through a PKC delta-dependent mechanism and that a sustained increase in intracellular free calcium is sufficient to induce and is required for granulosa cell apoptosis. Additional studies demonstrated that in spontaneously immortalized granulosa cells, basic fibroblast growth factor increased PKC delta activity by 60% within 2.5 min compared with serum-free control levels. Rottlerin attenuated basic fibroblast growth factor's ability to stimulate PKC delta activity and to maintain intracellular free calcium. Further, intracellular free calcium levels in spontaneously immortalized granulosa cells transfected with a PKC delta antibody in the presence of basic fibroblast growth factor were 2-fold higher than those spontaneously immortalized granulosa cells transfected with IgG. Similarly, transfecting spontaneously immortalized granulosa cells with a specific PKC delta-substrate increased intracellular free calcium compared with spontaneously immortalized granulosa cells transfected with a specific substrate for PKC epsilon. Moreover, basic fibroblast growth factor increased and rottlerin attenuated (45)Ca efflux by 50% compared with that in basic fibroblast growth factor-treated cells. Finally, an inhibitor of the plasma membrane calciumadenosine triphosphatase pump suppressed (45)Ca efflux, elevated intracellular free calcium, and induced apoptosis. Collectively, these studies demonstrate that basic fibroblast growth factor activates PKC delta, which, in turn, stimulates calcium efflux, accounting in part for basic fibroblast growth factor's ability to maintain calcium homeostasis and, ultimately, granulosa cell viability.
先前的研究表明，碱性成纤维细胞生长因子可防止颗粒细胞凋亡。以下六项观察结果有助于深入了解碱性成纤维细胞生长因子介导其抗凋亡作用的机制。首先，用细胞内钙螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸处理颗粒细胞，当颗粒细胞缺乏碱性成纤维细胞生长因子时可防止凋亡。其次，用毒胡萝卜素（一种已知可增加细胞内游离钙的试剂）处理，即使在存在碱性成纤维细胞生长因子的情况下也会诱导颗粒细胞凋亡。第三，蛋白激酶C（PKC）的激活剂可模拟碱性成纤维细胞生长因子的抗凋亡作用，而PKC抑制剂则可阻断该作用。第四，持续暴露于碱性成纤维细胞生长因子可维持细胞内游离钙水平相对恒定，而PKC抑制剂可导致细胞内游离钙持续升高2至3倍。第五，颗粒细胞以及自发永生化的颗粒细胞均显示表达PKCδ、-λ和-ζ。最后，PKCδ特异性抑制剂rottlerin可阻断碱性成纤维细胞生长因子在颗粒细胞和自发永生化颗粒细胞中的抗凋亡作用。这些研究表明，碱性成纤维细胞生长因子通过PKCδ依赖性机制调节细胞内游离钙，并且细胞内游离钙的持续升高足以诱导颗粒细胞凋亡且是其必需条件。进一步的研究表明，在自发永生化的颗粒细胞中，与无血清对照水平相比，碱性成纤维细胞生长因子在2.5分钟内可使PKCδ活性增加60%。rottlerin减弱了碱性成纤维细胞生长因子刺激PKCδ活性和维持细胞内游离钙的能力。此外，在存在碱性成纤维细胞生长因子的情况下，用PKCδ抗体转染的自发永生化颗粒细胞中的细胞内游离钙水平比用IgG转染的自发永生化颗粒细胞高2倍。同样，与用PKCε特异性底物转染的自发永生化颗粒细胞相比，用PKCδ特异性底物转染自发永生化颗粒细胞可增加细胞内游离钙。此外，与碱性成纤维细胞生长因子处理的细胞相比，碱性成纤维细胞生长因子使（45）Ca外流增加50%，而rottlerin则减弱了这种作用。最后，质膜钙-腺苷三磷酸酶泵的抑制剂可抑制（45）Ca外流、升高细胞内游离钙并诱导凋亡。总的来说，这些研究表明碱性成纤维细胞生长因子激活PKCδ，进而刺激钙外流，这部分解释了碱性成纤维细胞生长因子维持钙稳态以及最终维持颗粒细胞活力的能力。
