Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.
Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.
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DOI：
10.1210/mend.13.8.0334
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发表时间：
1999-08
影响因子：
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通讯作者：
I. GONZALEZ-ROBAYNA;Tamara Alliston;Patricia Buse;G. Firestone;J. S. Richards
中科院分区：
文献类型：
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作者：
I. GONZALEZ-ROBAYNA;Tamara Alliston;Patricia Buse;G. Firestone;J. S. Richards
颗粒细胞对FSH（cAMP）的反应性随着这些细胞从生长卵泡的增殖阶段转换到LH诱导的黄体化后的终末分化非增殖阶段而变化。为了分析这种转变，使用了两种良好表征的培养系统。1)将从未成熟大鼠分离的颗粒细胞培养在无血清培养基中，该系统允许分析对激素/cAMP的动态短期反应。2)在含有1%FBS的培养基中培养来自排卵前（PO）卵泡的颗粒细胞，这些卵泡已在体内暴露于hCG的激增浓度（PO/hCG），该系统允许分析经历与黄体化相关的不可逆的长期变化的细胞。为了分析cAMP反应性转换的生化基础，A-激酶途径组分的定位与两个cAMP靶基因，芳香化酶（CYP19）和血清和糖皮质激素诱导的激酶（Sgk）的表达有关。使用C亚基、RII α/β亚基、CREB（cAMP-调节元件结合蛋白）、磷酸化CREB、CBP（CREB结合蛋白）和Sgk的特异性抗体，通过Western印迹和间接免疫荧光分析A-激酶途径的组分。C亚基和CREB的细胞水平在所有细胞类型和激素处理中是相似的。CREB和CBP是核的; RII α/β局限于细胞质篮状结构。在未成熟颗粒细胞中加入FSH可在1h内引起C亚基的快速核输入。FSH后6小时，核C亚基减少，但在6、24或48小时加入毛喉素后，核C亚基可以迅速重新输入细胞核。核C亚基与磷酸化CREB的快速但短暂的增加有关。FSH以双相方式诱导Sgk，其中蛋白在1h时为核蛋白，在48h时为胞浆蛋白。芳香酶mRNA仅在FSH后24 - 48小时表达，磷酸二酯酶或磷酸酶不会改变这种模式。在黄体化（PO/hCG）颗粒细胞，免疫反应性C亚基定位在核中的点状图案以及细胞质篮状结构，分布模式不改变毛喉素。芳香化酶，Sgk和磷酸化CREB的表达水平升高，在非毛喉素反应的方式。最值得注意的是，磷酸化CREB和Sgk都优先定位在细胞质内的点状图案，而不改变毛喉素。总的来说，这些数据表明，当颗粒细胞分化为黄体细胞的亚细胞定位（细胞核与细胞质）的A-激酶通路组件的显着变化。因此，无论是核进口和出口的机制，还是不同的对接地点（和功能？）与终末分化的黄体细胞相比，A-激酶、磷酸化CREB和Sgk在颗粒细胞中的定位。
The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.