Ruh M F, Singh R K, Ruh T S, Shyamala G
Department of Physiology, St. Louis University School of Medicine, MO 63104.
J Steroid Biochem. 1987 Dec;28(6):581-6. doi: 10.1016/0022-4731(87)90383-9.
We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.
我们最近对小鼠乳腺雌激素受体（ER）与乳腺染色质受体位点的相互作用进行了表征，并证明来自雌激素抵抗性泌乳乳腺的ER不与染色质结合。在本研究中，我们对从小鼠未生育和泌乳小鼠分离的糖皮质激素受体的染色质结合进行了表征，以便更好地理解受体与染色质结合与组织类固醇生成敏感性之间的关系。乳腺染色质与纤维素共价连接，并依次用0 - 8 M盐酸胍脱蛋白。使用通过在DEAE - 纤维素柱上分级分离获得的部分纯化的[3H]地塞米松标记的糖皮质激素受体复合物（GR）来测定与完整染色质以及用盐酸胍脱蛋白的染色质的结合。来自未生育小鼠乳腺的[3H]GR与同一组织的染色质级分的结合显示，在用5 - 6 M盐酸胍预先提取的染色质上具有最大结合活性（受体位点）。[3H]GR的结合具有高亲和力（Kd = 0.2 nM）且可饱和。对从未生育小鼠乳腺分离的[3H]ER和[3H]GR的染色质结合模式的同时比较表明，用4 - 6 M盐酸胍提取获得的染色质亚级分包含[3H]ER和[3H]GR的受体位点；然而，虽然[3H]ER与4.5 M和5.5 M位点结合，但[3H]GR与5 M和6 M位点结合。竞争实验支持了染色质受体位点的类固醇受体特异性。因此，4 - 6 M染色质级分包含糖皮质激素受体和雌激素受体的不同受体位点。此外，我们的研究表明，从未生育和泌乳小鼠乳腺分离的[3H]GR与其同源染色质的结合模式基本相似。因此，与雌激素受体不同，泌乳乳腺的糖皮质激素受体能够有效地与染色质受体位点结合，这支持了我们之前的观点，即泌乳小鼠乳腺的雌激素不敏感性可能至少部分是由于ER与染色质受体位点的相互作用受阻。
