Maru Benit S, Tobias Jonathan H, Rivers Caroline, Caunt Christopher J, Norman Michael R, McArdle Craig A
Laboratory for Integrated Neurosciences and Endocrinology, University of Bristol, UK.
Bone. 2009 Jan;44(1):102-12. doi: 10.1016/j.bone.2008.09.016. Epub 2008 Oct 11.
SERMs act as ER agonists in bone despite their antagonistic properties in other tissues. As well as inhibiting bone remodelling, this effect may involve stimulation of osteoblast activity, in light of evidence from recent in vivo studies. However, progress in exploring this action has been hampered by a lack of accurate in vitro models. For example, ER antagonists are reported to stimulate reporter assays based on estrogen target genes in osteoblasts, contrary to their inhibitory effects in vivo. We examined whether evaluating global aspects of ER function provides a more accurate reflection of ER activation in osteoblasts, based on the use of morphological and/or transcriptional read-outs with green fluorescent protein (GFP)-receptor chimeras. Osteoblast-like (ROS and U2OS) and breast cancer (MCF7) cells were transfected with a human ERalpha-GFP fusion protein, and treated with ER agonists (17beta-estradiol, and dienestrol), antagonists (ICI 182,780 and ZK 164015) and SERMs (tamoxifen, raloxifene, 4-hydroxytamoxifen (4-HT) and hexestrol). We investigated cellular compartmentalisation of these constructs by fluorescence microscopy, nuclear mobility by fluorescence recovery after photobleaching (FRAP), and global activation of estrogenic transcription using a ERE-luc reporter. SERMs caused a modest increase in ERE-luc activity in osteoblast-like cells (but not in breast cells), and a reduction in nuclear mobility in breast (but not osteoblast-like) cells. These studies were then repeated using a GFP chimera where the human GR ligand binding domain (LBD) was replaced by the human ERalpha LBD (ERGR-GFP), combined with a GRE-luc reporter. Interestingly, SERMs increased both cytoplasmic to nuclear translocation of ERGR-GFP, and GRE-luc reporter activity, in osteoblast-like (but not breast) cells. Indeed, transcriptional responses to SERMs in osteoblast-like cells were considerably greater with the ERGR/GRE-luc than the ERalpha/ERE-luc system, 4-HT inducing 300 and 25% increases in reporter activity respectively. ER antagonists were entirely without effect. We conclude that evaluation of global estrogenic activity, as opposed to activation of a specific target gene, provides a more accurate read-out for osteoblast stimulation. In particular, ERGR-mediated GRE-luc activity provides a high signal response to estrogen agonists and SERMs, in a cell context dependent manner closely resembling that observed in vivo. Further studies utilising this system are justified to explore the mechanistic basis for estrogenic stimulation of osteoblast activity, and to identify newer SERMs capable of targeting this activity.
选择性雌激素受体调节剂（SERMs）在骨骼中表现为雌激素受体（ER）激动剂，尽管它们在其他组织中具有拮抗特性。除了抑制骨重塑外，根据最近体内研究的证据，这种作用可能涉及刺激成骨细胞活性。然而，由于缺乏准确的体外模型，探索这一作用的进展受到了阻碍。例如，据报道，ER拮抗剂在成骨细胞中会刺激基于雌激素靶基因的报告基因检测，这与它们在体内的抑制作用相反。我们基于使用绿色荧光蛋白（GFP）-受体嵌合体的形态学和/或转录读数，研究了评估ER功能的整体方面是否能更准确地反映成骨细胞中ER的激活情况。将人ERα-GFP融合蛋白转染到成骨样细胞（ROS和U2OS）和乳腺癌细胞（MCF7）中，并用ER激动剂（17β-雌二醇和己烯雌酚）、拮抗剂（ICI 182,780和ZK 164015）和SERMs（他莫昔芬、雷洛昔芬、4-羟基他莫昔芬（4-HT）和己烷雌酚）进行处理。我们通过荧光显微镜研究了这些构建体的细胞区室化，通过光漂白后荧光恢复（FRAP）研究了核迁移，并使用ERE-荧光素酶报告基因研究了雌激素转录的整体激活。SERMs在成骨样细胞中导致ERE-荧光素酶活性适度增加（但在乳腺癌细胞中没有），而在乳腺癌细胞（但不是成骨样细胞）中导致核迁移减少。然后使用GFP嵌合体重复这些研究，其中人糖皮质激素受体（GR）配体结合域（LBD）被人ERα LBD（ERGR-GFP）取代，并结合GRE-荧光素酶报告基因。有趣的是，SERMs在成骨样细胞（但不是乳腺癌细胞）中增加了ERGR-GFP从细胞质到细胞核的转位以及GRE-荧光素酶报告基因活性。事实上，与ERα/ERE-荧光素酶系统相比，成骨样细胞中对SERMs的转录反应在ERGR/GRE-荧光素酶系统中要大得多，4-HT分别导致报告基因活性增加300%和25%。ER拮抗剂完全没有作用。我们得出结论，与激活特定靶基因相反，评估整体雌激素活性为成骨细胞刺激提供了更准确的读数。特别是，ERGR介导的GRE-荧光素酶活性以细胞背景依赖的方式对雌激素激动剂和SERMs提供高信号反应，这与体内观察到的情况非常相似。利用该系统进行进一步研究是合理的，以探索雌激素刺激成骨细胞活性的机制基础，并鉴定能够靶向这种活性的新型SERMs。
