Fiorelli G, Gori F, Petilli M, Tanini A, Benvenuti S, Serio M, Bernabei P, Brandi M L
Department of Clinical Physiopathology, University of Florence, Italy.
Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2672-6. doi: 10.1073/pnas.92.7.2672.
The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.
雌激素雌二醇 - 17β（17β - E2）对骨骼的主要生物学作用是减少骨吸收。然而，17β - E2是直接影响破骨细胞分化或功能，还是通过其对成骨细胞的作用来影响，目前尚不清楚。为了研究这个问题，我们检测了人破骨细胞前体细胞系FLG 29.1中功能性雌激素受体（ERs）的证据。用针对人ER mRNA的32P标记cDNA探针进行逆转录 - PCR扩增产物的Southern印迹分析表明，FLG 29.1细胞表达ER mRNA。[3H]17β - E2与核ERs的结合具有类固醇特异性，每个细胞核约有400个可饱和的高亲和力（Kd约为1 nM）结合位点。用[3H]他莫昔芬氮丙啶共价标记的核ERs经SDS/PAGE和用人ER的D75单克隆抗体进行Western印迹分析，显示表观分子量为65,000。用0.1、1.0或10 nM 17β - E2预处理细胞可诱导孕酮与FGL 29.1细胞的剂量和时间依赖性特异性结合，用10 nM和100 nM 17β - E2刺激细胞可显著（P < 0.05）降低细胞增殖。通过用含有卵黄蛋白原A2基因的雌激素反应元件和细菌氯霉素乙酰转移酶报告基因的pERE - BLCAT质粒瞬时转染细胞来检测ER基因的转录活性。与对照组相比，用10 nM 17β - E2处理FLG 29.1细胞可使氯霉素乙酰转移酶表达增加5至29倍。这些观察结果表明雌激素在破骨细胞生成中可能发挥作用。
