Potential use of an estrogen–glucocorticoid receptor chimera as a drug screen for tissue selective estrogenic activity Benit S. Maru a , Jonathan H. Tobias b, ⁎, Caroline Rivers a , Christopher J. Caunt a , Michael R. Norman a , Craig A. McArdle a a Laboratory for Integrated Neurosciences and Endocrinology, University of Bristol, UK b Academic Rheumatology, University of Bristol, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK abstract article info Article history: Received 9 July 2008 Revised 24 September 2008 Accepted 29 September 2008 Available online 11 October 2008 Edited by: J. Aubin Keywords: SERMs Estrogen receptor alpha Osteoblasts Estradiol Reporter assay 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 ERα–GFP fusion protein, and treated with ER agonists (17β-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 ERα 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 ERα/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. © 2008 Elsevier Inc. All rights reserved. Introduction As well as suppression of osteoclastic bone resorption, in vitro and in vivo studies suggest beneficial effects of SERMs on bone involve stimulation of osteoblastic bone formation [1–3]. Although clinical evidence for a positive effect of SERMs on bone formation is currently lacking, such a mode of action seems likely given that several clinical studies suggest that estrogen directly stimulates osteoblast activity in humans [4]. To provide a basis for exploring the mechanism of action of SERM-dependent stimulation in osteoblasts, and for screening newer more effective compounds, we previously examined whether reporters based on osteoblast-regulatory genes such as bone morphogenetic protein (BMP)-6 can be used to evaluate preferential responses to SERMs in osteoblasts. We found that SERMs, but not 17β-estradiol (E2), stimulated BMP-6 reporter activity in ROS (osteosarcoma) cells transiently transfected with ERα, whereas no stimulation was observed in MCF7 (breast cancer) cells [5]. Similar findings have been reported for other putative osteoblast target genes such as BMP-4, TGFβ3 and Cbfa1 [5–8]. However, the physiological relevance of these reporters can be brought into question, as they also appear to be activated by anti- estrogens such as ICI 182,780 [5–8], which we previously found produces a pronounced inhibitory effect on bone formation in vivo [9]. It has been suggested that binding of SERMs to ERs induces (or stabilises) receptor conformations that differ from those induced by native ligands such as E2, facilitating interactions with different co- Bone 44 (2009) 102–112 ⁎ Corresponding author. Fax: +44117 959 5936. E-mail address: Jon.Tobias@bristol.ac.uk (J.H. Tobias). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.09.016 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone