Restrain of bone growth by Estrogen-Mimetic Peptide-1 (EMP-1): A micro-computed tomographic study Roni Kasher a, *, Alon Bajayo b , Yankel Gabet b , Nava Nevo c , Mati Fridkin d , Ephraim Katchalski-Katzir e , Fortune Kohen c , Itai Bab b a Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, The Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus 84990, Israel b Bone Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel c Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel d Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel e Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel 1. Introduction Estrogen is a key regulator of skeletal mass. Its depletion through menopause or ovariectomy (OVX) leads to bone loss that can be prevented by estrogen replacement therapy. The effects of estrogen are mediated by the estrogen receptors ERa and ERb which function as ligand-activated transcriptional factors [14]. These receptors exhibit different binding affinities for endogenous, synthetic and naturally occurring ligands [18]. Each receptor is expressed at different levels in various cell types [1,3,8,15]. ER activation is involved in the regulation of longitudinal [6,24] and radial bone growth [32]. We have recently developed synthetic peptides with estrogen- like activity [13,33] as potential lead compounds for new ER ligands that do not share the adverse effects of currently available estrogen-based therapies. Using a strategy that com- bines (i) an anti-estradiol monoclonal antibody (mAb-E2) as a bait for screening a phage-display peptide library [33]; (ii) Systematic- Residue Replacement, a new method for peptide affinity optimization [12], we identified a linear hexapeptide, VSWFFE that we named Estrogen-Mimetic Peptide 1 (EMP-1) [13]. EMP-1 binds mAb-E2 with high affinity (IC 50 = 0.006 mM). It inhibits the binding of 3 H-estradiol to both ERa and ERb at medium potency (IC 50 = 100 mM) and possesses estrogen-like activity in vitro such as stimulation of DNA synthesis and transcriptional activity in breast cancer cells transfected with an ER element- luciferase construct. In vivo in the uterus, EMP-1 stimulates expression of the progesterone receptor, an estrogenic res- ponse gene [17]. In addition, in bone and several soft tissues it enhances the activity of creatine kinase, an estrogen responsive enzyme, an effect that is abolished by the selective ER antagonist raloxifene [13]. The present study was undertaken to test potential estrogen- like activities of EMP-1 in mouse bone, in the presence and absence of estrogen. EMP-1 failed to prevent OVX-induced bone loss. Surprisingly, it enhanced the age-related diminution in growth plate thickness and inhibited longitudinal bone growth. These effects occurred only in the absence of estrogen. Peptides 30 (2009) 1181–1186 ARTICLE INFO Article history: Received 10 November 2008 Received in revised form 26 February 2009 Accepted 26 February 2009 Available online 14 March 2009 Keywords: Estrogen-Mimetic Peptide Bone growth Micro-computed tomography Estrogenic activity ABSTRACT Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (mCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new mCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients. ß 2009 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +972 8 6563531; fax: +972 8 6563503. E-mail address: kasher@bgu.ac.il (R. Kasher). Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.02.019