一個多脯胺酸酸性蛋白在子宮內膜細胞的轉錄調控和加強該細胞對雌性素反應的能力

Abstract

利用二維電泳比較由人工合成己烯雌酚 (DES, diethylstilbesterol) 誘導幼鼠所產生之子宮液與正常成鼠於動情週期所產生之子宮液兩者蛋白質的成分，發現一個分子量為 14 kDa 、等電點為 4.6 的蛋白於成鼠表現，但於 DES 誘導的幼鼠沒有表現。由 MALDI-Q-TOF 質譜與 N端分析，證實此一蛋白質為 proline-rich acidic protein (PRAP)。 檢視成鼠性腺，Prap mRNA僅於子宮內膜細胞表現。於動情週期中， Prap mRNA從動情前期 (proestrus) 開始表現，於動情期 (estrus) 表現明顯增加，至早期動情後期 (metestrus I) 達到最高峰，於後期動情後期 (metestrus II) 快速遞減，最後於動情間期 (diestrus) 則幾乎沒有表現。 對未成熟的小鼠施予雌性素 (17 β-estradiol) (0.01- 10 μg/g body weight) 三天後，皆於子宮中檢視到Prap mRNA 與PRAP蛋白質的表現，但表現量隨著雌性素劑量增多而減少。單獨施打黃體素 (progesterone) 並不引發Prap 的表現，共同施打雌性素和黃體素，並沒有抑制雌性素的刺激。進一步證實Prap 受雌性素的刺激和雌性素α亞型有關。三週大幼鼠施予連續三天DES (0.1 μg/g body weight) 誘導的子宮液沒有發現PRAP。若先施打DES於未成熟小鼠，待其年齡到達成熟週數，Prap 基因在子宮的表現，相對於正常成鼠也顯著減少。 重組PRAP 蛋白外加於培養液會內吞至人類子宮內膜腺癌細胞 (Ishikawa cells)，雌性素會提高人類子宮內膜腺癌細胞的鹼性磷酸酶酵素活性及其反應基因，單獨外加重組PRAP 蛋白並無影響，若同時加入雌性素和PRAP， PRAP 加強雌性素所刺激的鹼性磷酸酶酵素活性及其反應基因。

Mice were used as experimental animals to study the functional role of uterine PRAP and how its expression is regulated by ovarian steroids. Soluble proteins in the uterine luminal fluid (ULF) of estrous females were resolved by 2-D gel electrophoresis. The protein spot corresponding to PRAP on the gel slab was identified by proteomic analysis. However, PRAP was absent in the ULF of DES- treated immature mice. Among the sexual glands of adults, both PRAP and its transcript were exclusively expressed in the endometrial epithelium of estrous females. Uterine Prap expression changed during the estrous cycle, in which the transcript levels progressively increased from proestrus via estrus to early metestrus, after which they rapidly declined via late metestrus to a very low level in diestrus. Daily injection of estradiol (E2) at a dose of 0.1 μg•g-1•day-1 to immature females for 3 consecutive days markedly stimulated uterine Prap transcription, whereas transcription was not initiated in the animals that received an equal dose of diethylstilbestrol dipropionate (DES). Relative to the normal estrus female, prepubertal DES exposure decreases remarkedly in the uterine Prap expression when the animals became sexually matured. The E2-stimulated Prap transcription involved the α-type estrogen receptor; at higher E2 doses, a lower level of uterine Prap mRNA and a lower amount of PRAP protein in ULF were detected. Using the Ishikawa cell line as a surrogate model of endometrial epithelium, we demonstrated that exogenous PRAP in the milieu rapidly internalizes into the cells to enhance endometrial estrogen responsiveness.