Human osteoclasts express oxytocin receptor Silvia Colucci, * Graziana Colaianni, Giorgio Mori, Maria Grano, and Alberta Zallone Department of Human Anatomy and Histology, University of Bari, Policlinico-P.zza G. Cesare 11, Bari 70 124, Italy Received 22 July 2002 Abstract Increasing evidences demonstrated many new targets for the hypothalamic hormone oxytocin, as the regulation of food balance and in some cases of leptin secretion. Considering that leptin is a potent inhibitor of bone formation and that oxytocin receptors (OTR) were detected in normal human osteoblasts, we investigated if OTR was expressed by human osteoclasts (hOCs) and the effect of the hormone on these cells. Here, we demonstrate by immunofluorescence and by Western blot analysis the expression of OTR by fully differentiated hOCs and by their precursors (pOCs). We also show that the receptor is functional, as OT treatment induces an increase of ½Ca 2þ  i , and that the hormone may affect osteoclastogenesis, since it increases the number of pre-osteo- clasts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Osteoclasts; Pre-osteoclasts; Oxytocin; Oxytocin receptor A large body of evidences recently suggest a central, neuroendocrine regulation of bone remodeling [1], identifying leptin as a potent inhibitor of bone forma- tion acting through the central nervous system [2,3]. On the other hand, new findings show a possible role for oxytocin (OT), a neurohypophysial peptide hormone, in theregulatorycontrolofleptin[4,5]andithasalsobeen very recently published that leptin may inhibit the se- cretion of OT [6]. Foralongtime,theOTwasknowntoexertitseffect on uterine smooth muscle contraction during labor and milk ejection during lactation [7,8], successively detected in both female and male, and further reported to be implicated in neuroadaptive processes exerting a wide spectrum of central and peripheral effects [9,14]. All these roles are mediated by specific cell surface receptor (OTR) detected in different organs, tissues, and tumor cells [15,20] and recently found in osteosarcoma cell lines [21] as well as in normal human osteoblasts [22]. Thus, on the basis of these data, here we investigated whether completely differentiated human osteoclasts (hOCs) and their precursors, obtained from peripheral blood mononuclear cells (PBMCs), express OTR, if the receptor was functional and the possible effect of the hormoneonthesecells.OurresultsindicatethatOTRis expressedbyhumanosteoclastsandtheirprecursorsand that the hormone controls osteoclastogenesis increasing the number of osteoclast precursors. Materials and methods Cell cultures Human osteoclasts. Osteoclast precursors were obtained from heparinized human peripheral blood mononuclear cells (PBMCs) collectedfromhealthywomandonorsfrom25to35yearsold.PBMCs were isolated by centrifugation over Histopaque 1077 (Sigma Chemi- cal, St. Louis, MO) density gradients, washed, and diluted at 1:3  10 6 cells/ml in a-minimal essential medium (a-MEM) supple- mented with 10% fetal bovine serum (FBS, Gibco, Uxbridge, UK), 100IU/ml penicillin, 100 lg/ml streptomycin, 2:5 lg/ml amphotericin B,and50IU/mlmycostatin(Gibco,Uxbridge,UK).PBMCswerethen culturedfor5–7daysinthepresenceof100ng/mlrecombinanthuman macrophagecolonystimulatingfactor(h-MCSF)(R&DSystems,MN, USA). The obtained cells were after detached with 10mM EDTA, dilutedatadensityof5–10  10 5 cells/cm 2 ,seededontopetridishesor coverslips, and cultured in a-MEM in the presence of 10% FBS, 100ng/ml MCSF, and 50ng/ml receptor activator of NF-jB ligand (RANKL) (R&D Systems, MN, USA) to obtained fully differentiated human osteoclasts (OCs). Culture medium was replaced every three dayswithfreshmediumsupplementedwiththeagentsdescribedabove, multinucleated and tartrate resistant acid phosphatase (TRAP) posi- tive cells appeared in culture from day 10 [23]. Biochemical and Biophysical Research Communications 297 (2002) 442–445 www.academicpress.com BBRC * Corresponding author. Fax: +39-080-547-8306. E-mail address: s.colucci@anatomia.uniba.it (S. Colucci). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02009-0