Rivera-Gonzalez R, Petersen D N, Tkalcevic G, Thompson D D, Brown T A
Department of Molecular Sciences, Pfizer Central Research, Groton, CT 06340, USA.
J Steroid Biochem Mol Biol. 1998 Jan;64(1-2):13-24. doi: 10.1016/s0960-0760(97)00142-8.
The identification and characterization of estrogen regulated genes in reproductive tissues is an important step in understanding estrogen's mechanism of action in sexual development and neoplasia. It is also important, given the clinical interest, to evaluate the molecular effects of estrogen agonists/antagonists such as tamoxifen and droloxifene in reproductive tissues. In this report, our goal was to identify estrogen regulated genes in the uterus and to compare the regulation by estrogen and tamoxifen with that of droloxifene. A subtractive cDNA library strategy was developed to identify estrogen-regulated genes in the uteri of ovariectomized rats 4 h after treatment with 17-alpha-ethynyl estradiol (30 microg/kg). The mRNAs encoding 8 genes were confirmed by Northern blot analysis to be induced at early times following estrogen administration. Calcium binding protein 9 kDa and complement protein 3 are well characterized estrogen regulated genes that were identified in the library and served as markers for estrogen action. In addition, mRNAs encoding the interleukin 4 receptor, heat-shock protein 70 kDa, metallothionein, tumor necrosis factor regulated gene 6, inositol-1-monophosphate synthase, and cyr-61 were induced in the uterus by estrogen. The identified mRNAs were then examined for regulation by droloxifene (1 and 10 mg/kg, p.o.) and tamoxifen (10 mg/kg, p.o.). Both droloxifene and tamoxifen induced mRNA levels for all of these genes. However, clear quantitative and temporal differences were observed when comparing estrogen versus droloxifene versus tamoxifen. For example, estrogen induced IL4 receptor mRNA to a greater degree than did tamoxifen or droloxifene. Conversely, tamoxifen resulted in a much greater induction of cyr61 than did either estrogen or droloxifene. Droloxifene at 1 mg/kg, an efficacious dose for prevention of bone loss in this model, did not or only slightly induced the mRNA for all of the genes examined with the exception of cyr61. In conclusion, the modified subtractive library method used in this study proved to be efficient in the identification of estrogen-regulated genes in the uterus. The identities of the regulated genes were consistent with the concept that estrogen functions to prime uterine tissue for increased responsivity to extracellular signals such as growth factors and cytokines. Elucidating the physiological role of these newly identified estrogen responsive genes and the mechanisms responsible for the different responses to droloxifene versus estrogen and tamoxifen may be important in enhancing our understanding of tissue selective estrogen agonists/antagonists.
鉴定和表征生殖组织中雌激素调节基因是了解雌激素在性发育和肿瘤形成中作用机制的重要一步。鉴于临床兴趣，评估雌激素激动剂/拮抗剂（如他莫昔芬和屈洛昔芬）在生殖组织中的分子效应也很重要。在本报告中，我们的目标是鉴定子宫中雌激素调节基因，并比较雌激素、他莫昔芬和屈洛昔芬的调节作用。我们开发了一种消减cDNA文库策略，以鉴定用17-α-乙炔基雌二醇（30μg/kg）处理4小时后的去卵巢大鼠子宫中雌激素调节的基因。通过Northern印迹分析证实，编码8个基因的mRNA在雌激素给药后的早期被诱导。钙结合蛋白9 kDa和补体蛋白3是在文库中鉴定出的特征明确的雌激素调节基因，并作为雌激素作用的标志物。此外，雌激素在子宫中诱导了编码白细胞介素4受体、热休克蛋白70 kDa、金属硫蛋白、肿瘤坏死因子调节基因6、肌醇-1-单磷酸合酶和cyr-61的mRNA。然后检测鉴定出的mRNA受屈洛昔芬（1和10 mg/kg，口服）和他莫昔芬（10 mg/kg，口服）的调节情况。屈洛昔芬和他莫昔芬均诱导了所有这些基因的mRNA水平。然而，在比较雌激素与屈洛昔芬与他莫昔芬时，观察到明显的定量和时间差异。例如，雌激素诱导IL4受体mRNA的程度比他莫昔芬或屈洛昔芬更大。相反，他莫昔芬诱导cyr61的程度比雌激素或屈洛昔芬都大得多。在该模型中对预防骨质流失有效的1 mg/kg屈洛昔芬，除cyr61外，对所有检测基因均未诱导或仅轻微诱导其mRNA。总之，本研究中使用的改良消减文库方法被证明在鉴定子宫中雌激素调节基因方面是有效的。所调节基因的身份与雌激素作用是使子宫组织对细胞外信号（如生长因子和细胞因子）的反应性增加的概念一致。阐明这些新鉴定的雌激素反应性基因的生理作用以及对屈洛昔芬与雌激素和他莫昔芬不同反应的机制，可能对增强我们对组织选择性雌激素激动剂/拮抗剂的理解很重要。
