Aharoni D, Dantes A, Amsterdam A
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Endocrinology. 1993 Sep;133(3):1426-36. doi: 10.1210/endo.133.3.7689957.
Steroidogenesis in granulosa cells can be stimulated by gonadotropic hormones and substances elevating cAMP. This cAMP-dependent metabolic event can be enhanced by peptide growth factors such as insulin, insulin-like growth factor, and epidermal growth factor, but the mechanism of cooperation between these two different signaling pathways is not yet clear. We have tested whether enhancement of tyrosine phosphorylation by vanadate, which blocks tyrosine phosphatases, is able to mimic the effect of growth factors on cAMP-induced steroidogenesis and investigated the cellular components involved in such modulation. Ortho- and metavanadate at 0.1-1.0 mM, when added to primary granulosa cell cultures, stimulated by gonadotropic hormones or forskolin, enhanced progesterone production by 1.5- to 9.0-fold within 120 min. Pervanadate showed a similar effect on steroidogenesis at a concentration one order of magnitude lower than ortho- or meta-vanadate. Phenylarsine-oxide, another blocker of tyrosine phosphatase, stimulated forskolin-induced steroidogenesis by 2.5-fold at 30 microM. In contrast, okadaic acid and calyculin A, which block specifically serine and threonine phosphatase, had no effect on steroidogenesis, when used at concentrations of 1 microM and 10 nM, respectively. The stimulation by vanadate was associated with a pronounced change in cell shape and total collapse of the actin network, which retracts to form a few large actin aggregates of 1-7 microns in diameter in the perinuclear region as revealed by visualization of actin by rhodamine-phalloidin staining under the fluorescent microscope. Steroidogenesis is not affected in cells treated with vanadate alone; the effect of vanadate on the actin cytoskeleton is much less pronounced. Electron microscopy of ultra-thin sections showed massive breakdown of thin filament cables in cells stimulated with vanadate together with gonadotropic hormone or forskolin. Massive clustering of lipid droplets and mitochondria as well as sharp increase in the electron-density of mitochondrial matrix was also observed in the stimulated cells. The action of vanadate in cAMP-stimulated cells leads to massive tyrosine phosphorylation of intracellular proteins in the range of 22-200 kilodaltons. It is suggested that the cross-talk between the cAMP pathway and tyrosine phosphorylation, which leads to enhanced steroidogenesis may be mediated by phosphorylation of cytoskeleton or associated proteins. The marked changes in lipid droplet-mitochondria interaction suggests that this enhanced steroidogenesis is due in part to mobilization of cholesterol into mitochondria in cells costimulated with vanadate and gonadotropins.
促性腺激素和能提高环磷酸腺苷（cAMP）水平的物质可刺激颗粒细胞中的类固醇生成。胰岛素、胰岛素样生长因子和表皮生长因子等肽类生长因子可增强这种依赖cAMP的代谢过程，但这两种不同信号通路之间的协同机制尚不清楚。我们测试了通过钒酸盐（可阻断酪氨酸磷酸酶）增强酪氨酸磷酸化是否能够模拟生长因子对cAMP诱导的类固醇生成的影响，并研究了参与这种调节的细胞成分。当向原代颗粒细胞培养物中添加0.1 - 1.0 mM的原钒酸盐和偏钒酸盐时，在促性腺激素或福斯高林的刺激下，120分钟内孕酮产量提高了1.5至9.0倍。过氧钒酸盐在比原钒酸盐或偏钒酸盐低一个数量级的浓度下对类固醇生成表现出类似的作用。另一种酪氨酸磷酸酶抑制剂苯砷氧化物在30 microM时可使福斯高林诱导的类固醇生成增加2.5倍。相比之下，分别以1 microM和10 nM的浓度使用时，特异性阻断丝氨酸和苏氨酸磷酸酶的冈田酸和花萼海绵诱癌素A对类固醇生成没有影响。钒酸盐的刺激与细胞形状的显著变化以及肌动蛋白网络的完全塌陷有关，在荧光显微镜下用罗丹明 - 鬼笔环肽染色观察肌动蛋白时发现，肌动蛋白网络收缩形成一些直径为1 - 7微米的大肌动蛋白聚集体，位于核周区域。单独用钒酸盐处理的细胞中类固醇生成不受影响；钒酸盐对肌动蛋白细胞骨架的影响要小得多。超薄切片的电子显微镜观察显示，在用钒酸盐与促性腺激素或福斯高林共同刺激的细胞中，细丝束大量断裂。在受刺激的细胞中还观察到脂滴和线粒体大量聚集以及线粒体基质电子密度急剧增加。钒酸盐在cAMP刺激的细胞中的作用导致细胞内22 - 200千道尔顿范围内的蛋白质大量酪氨酸磷酸化。有人提出，cAMP途径与酪氨酸磷酸化之间的相互作用导致类固醇生成增强，这可能是由细胞骨架或相关蛋白的磷酸化介导的。脂滴 - 线粒体相互作用的显著变化表明，这种增强的类固醇生成部分归因于在钒酸盐和促性腺激素共同刺激的细胞中胆固醇向线粒体的转运。
